[Histonet] Safranin O/ Fast green - protocol without alcohol
Histonetters! Our go-to stain is SafO for any bone/cartilage, but we always have the option to use toluidine or alcian blue. Just going out on a limb, but has anyone stained tissues with SafO/Fast Green, without using any alcohols after the SafO step? These samples have some issues when put into alcohol and if we wash the SafO in water, instead of alcohol, the stain isn't correct. What is it about the alcohol step after SafO that makes it work so well? And why does water cause such a problem? Next week, I will try a lower alcohol percentage after the SafO step to see how that works. Thanks, Merissa ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Safranin O cartilage staining
Brett Safranin O can be a bit tricky at times. There are a few tips that we have used through the years. 1. Fresh reagents are key. 2. For proteoglycan staining you can cut the sections a bit thicker and get better staining we typically cut the joint sections for Saf O at 6 to 7 microns in thickness, there are papers out there that recommend up to 8 microns in thickness. 3. Limit excess time in decal for some reason this particular stain may not work as well if the samples are in decal for an extended period of time, we have not seen this with toluidine blue which is another stain for proteoglycan. 4. We increase times in the safranin O reagent on occasion for the murine joints. Good Luck Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Laboratory Manager Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 Work (303) 682-3949 Fax (303) 682-9060 Cell (303) 881-0763 l...@premierlab.com www.premierlab.com Ship to address: 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Brett Tonkin Sent: Tuesday, May 21, 2013 11:08 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Safranin O cartilage staining Hi, We're having trouble staining murine articular cartilage with Safranin O. We first started using this stain last year and it was working nicely, with strong staining of both the articular cartilage and growth plate. After a while, the stain stopped working, with staining only visible in the growth plate. We replaced all solutions (including c/stain of fast green) and the staining worked. Yet again, it has stopped working, and this is only the second time the solutions have been used. Has anyone come across this before? Any help or advice would be greatly appreciated! Brett Tonkin Research Assistant Arthritis Research Laboratory Bone Cell Biology and Disease Unit St. Vincent's Institute Fitzroy, Victoria ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Safranin O cartilage staining
Hi, We're having trouble staining murine articular cartilage with Safranin O. We first started using this stain last year and it was working nicely, with strong staining of both the articular cartilage and growth plate. After a while, the stain stopped working, with staining only visible in the growth plate. We replaced all solutions (including c/stain of fast green) and the staining worked. Yet again, it has stopped working, and this is only the second time the solutions have been used. Has anyone come across this before? Any help or advice would be greatly appreciated! Brett Tonkin Research Assistant Arthritis Research Laboratory Bone Cell Biology and Disease Unit St. Vincent's Institute Fitzroy, Victoria ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Safranin O staining
Victor We process these samples on the tissue processor and not manually. You can use a kimwipe but we prefer the nylon tissue bags (the small ones 30 x 50 mm), they are a bit more expensive but they work well for us. We receive the pellets in a conical tube, we add a bit of eosin, about a ml, to the formalin in the tube and let it sit for about 10 minutes. We then use disposable pipets to retrieve the pellet from the conical tube, we don't use forceps at this stage, we draw up the pellet into the conical tube, open the tissue bag and then squirt the solution and pellet into the bag. Its helpful to have the pellet stained with eosin this way you can actually see it. We fold the bag and place it into the cassette. We embed in the pellets in the 15x15 mm base molds. We capture 4 levels per slide for a total of 8 sections, since the samples are so tiny we collect all the tissue onto unstained slides, this allows us to have sections through the pellet. What we do is collect several ribbions of tissue with about 15-20 sections per ribbon and then pick up 2 sections at a time. I will attach a diagram from our technical manual to help out in another e-mail. I'll see if I can get access to the paper. I can't comment on how to get rid of the debris since we do not perform that process. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.commailto:l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: Victor Wong [vhlw...@yahoo.com] Sent: Monday, February 18, 2013 8:37 PM To: Elizabeth Chlipala; Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Hi Liz, Thank you for your response and your invaluable experience in processing such samples. I'll try manual processing on next batch of samples. I am new to handle these samples. There would be a lot of cell debris during culture mixing with small pellet and this may be much bigger than the pellet. May I know how to get rid of it? Someone suggested to stain the pellet with eosin and place it in folded Kimwipe for processing. Is routine automatic staining helpful as we don't have a oven to melt paraffin nearby? We need to take a 30-minute walk to the lab for processing. BTW, I don't have access right to the paper. Anyway, the staining pictures on the website are very nice. Once, thank you for your help. Best Regards, Victor From: Elizabeth Chlipala l...@premierlab.com To: Victor Wong vhlw...@yahoo.com; Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:37 PM Subject: RE: [Histonet] Safranin O staining We have processed and stained these types of samples before. We do not embed the pellets in agarose prior to processing. We dye them with eosin and place them in a tea bag and process on a very short cycle - 10 minutes per station, embed section and stain. We have stained with safranin O, alcian blue, toluidine blue and IHC for collagen II. The safranin O comes out just fine as well as the others. We just receive the samples so I do not know anything about the preparation, collection and fixation. It possibly could be your construct, we have processed hundreds of these samples and the staining intensity and patterns do change. I expect this is due to the type of preparation. If you want to see pictures of the staining, you can go to this website: http://bioreagents.lifemapsc.com/products/purestem-chondropkg-4d20-8 or this paper: Sternberg H, Murai JT, Erickson IE, Funk WD, Das S, Wang Q, Snyder E, Chapman KB, Vangsness CT Jr, West MD. 2012 A human embryonic stem cell-derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme. Regen Med. 7(4):481-501 Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.commailto:l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong [vhlw...@yahoo.commailto:vhlw...@yahoo.com] Sent: Monday, February 18, 2013 12:35 AM To: Tony Henwood (SCHN); histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Dear Tony, Thank you for your prompt reply and the paper. I do fix the cell before processing in agarose and after embedding in agarose. It is inevitable to process in wax
Re: [Histonet] Safranin O staining
Hi Liz, Get it. Thank you for the detailed procedures. Best Regards, Victor From: Elizabeth Chlipala l...@premierlab.com To: Victor Wong vhlw...@yahoo.com; Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Wednesday, February 20, 2013 12:15 AM Subject: RE: [Histonet] Safranin O staining Victor We process these samples on the tissue processor and not manually. You can use a kimwipe but we prefer the nylon tissue bags (the small ones 30 x 50 mm), they are a bit more expensive but they work well for us. We receive the pellets in a conical tube, we add a bit of eosin, about a ml, to the formalin in the tube and let it sit for about 10 minutes. We then use disposable pipets to retrieve the pellet from the conical tube, we don't use forceps at this stage, we draw up the pellet into the conical tube, open the tissue bag and then squirt the solution and pellet into the bag. Its helpful to have the pellet stained with eosin this way you can actually see it. We fold the bag and place it into the cassette. We embed in the pellets in the 15x15 mm base molds. We capture 4 levels per slide for a total of 8 sections, since the samples are so tiny we collect all the tissue onto unstained slides, this allows us to have sections through the pellet. What we do is collect several ribbions of tissue with about 15-20 sections per ribbon and then pick up 2 sections at a time. I will attach a diagram from our technical manual to help out in another e-mail. I'll see if I can get access to the paper. I can't comment on how to get rid of the debris since we do not perform that process. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: Victor Wong [vhlw...@yahoo.com] Sent: Monday, February 18, 2013 8:37 PM To: Elizabeth Chlipala; Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Hi Liz, Thank you for your response and your invaluable experience in processing such samples. I'll try manual processing on next batch of samples. I am new to handle these samples. There would be a lot of cell debris during culture mixing with small pellet and this may be much bigger than the pellet. May I know how to get rid of it? Someone suggested to stain the pellet with eosin and place it in folded Kimwipe for processing. Is routine automatic staining helpful as we don't have a oven to melt paraffin nearby? We need to take a 30-minute walk to the lab for processing. BTW, I don't have access right to the paper. Anyway, the staining pictures on the website are very nice. Once, thank you for your help. Best Regards, Victor From: Elizabeth Chlipala l...@premierlab.com To: Victor Wong vhlw...@yahoo.com; Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:37 PM Subject: RE: [Histonet] Safranin O staining We have processed and stained these types of samples before. We do not embed the pellets in agarose prior to processing. We dye them with eosin and place them in a tea bag and process on a very short cycle - 10 minutes per station, embed section and stain. We have stained with safranin O, alcian blue, toluidine blue and IHC for collagen II. The safranin O comes out just fine as well as the others. We just receive the samples so I do not know anything about the preparation, collection and fixation. It possibly could be your construct, we have processed hundreds of these samples and the staining intensity and patterns do change. I expect this is due to the type of preparation. If you want to see pictures of the staining, you can go to this website: http://bioreagents.lifemapsc.com/products/purestem-chondropkg-4d20-8 or this paper: Sternberg H, Murai JT, Erickson IE, Funk WD, Das S, Wang Q, Snyder E, Chapman KB, Vangsness CT Jr, West MD. 2012 A human embryonic stem cell-derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme. Regen Med. 7(4):481-501 Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong [vhlw...@yahoo.com] Sent: Monday, February 18, 2013 12:35 AM To: Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Dear Tony, Thank you for your prompt
Re: [Histonet] Safranin O staining
Believe it or not, not because you are dealing with cells (small as they are) they require longer than 1 hour to be correctly fixed. This is what I would do: fix the cells for 8 hours minimum → prepare the pellet in agarose → process to paraffin (FFPE) block → section as usual. Now, check the distaining protocol, consult any histotechnique book because I think 2 min is Weigert is a very short time (remember the mordant) → try different staining times, or follow a known protocol (you can find that in either Peter Gray's or Lillie's books). René J. From: Victor Wong vhlw...@yahoo.com To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Sunday, February 17, 2013 9:34 PM Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O staining
Victor, I completely agree with Tony and Renee's methodology assessment but I would also ask you to look at your induced chondrogenesis model (I've never done this but have done many, many other cell culture models). If you go to this paper http://biotech.korea.ac.kr/bk21/bbs/upload/bk21bbs_cur_165_0.pdf in Tissue Engineering they describe in detail exactly what you are trying to do, if I read your question correctly. Their pictures of safrinin 0 staining of pelleted cells from culture could be described as light staining if you compare them to the red/orange color what we all know safranin o looks like on articular surfaces. I think that is a POOR positive control for their experiments but that's just me. In-vitro cultured cells prepared for histology will seldom stain the same as in-vivo material prepared for histology. And they had to hit cells pretty hard, 5ng TGF to see some staining. Have no idea what your cell culture methodologies are but what I think you are doing is all in that paper and you can see the results in the Safranin o pictures. There are ways to get better, more similar, positive staining controls then they used. Ray Koelling Research Scientist University of Washington Seattle - Original Message - From: Victor Wong vhlw...@yahoo.com To: histonet@lists.utsouthwestern.edu Sent: Sunday, February 17, 2013 6:34:12 PM Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Safranin O staining
We have processed and stained these types of samples before. We do not embed the pellets in agarose prior to processing. We dye them with eosin and place them in a tea bag and process on a very short cycle - 10 minutes per station, embed section and stain. We have stained with safranin O, alcian blue, toluidine blue and IHC for collagen II. The safranin O comes out just fine as well as the others. We just receive the samples so I do not know anything about the preparation, collection and fixation. It possibly could be your construct, we have processed hundreds of these samples and the staining intensity and patterns do change. I expect this is due to the type of preparation. If you want to see pictures of the staining, you can go to this website: http://bioreagents.lifemapsc.com/products/purestem-chondropkg-4d20-8 or this paper: Sternberg H, Murai JT, Erickson IE, Funk WD, Das S, Wang Q, Snyder E, Chapman KB, Vangsness CT Jr, West MD. 2012 A human embryonic stem cell-derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme. Regen Med. 7(4):481-501 Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong [vhlw...@yahoo.com] Sent: Monday, February 18, 2013 12:35 AM To: Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Dear Tony, Thank you for your prompt reply and the paper. I do fix the cell before processing in agarose and after embedding in agarose. It is inevitable to process in wax at higher than 45C as it is set by the routine workers. It is the first time I worked on cell block precessing. Do you have protocol of processing chondrogenic pellet by tissue processor? Any adjustment to my procesing and staining protocol? I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed. Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat? Best Regards, Victor From: Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au To: 'Victor Wong' vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:45 AM Subject: RE: [Histonet] Safranin O staining Victor, Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136). I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong Sent: Monday, 18 February 2013 1:34 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance
RE: [Histonet] Safranin O staining
Hi Victor, I suppose the take home message from Russ's paper (God rest his soul - we do miss him) is that heat will affect some stains so extrapolating to routine processing temperatures, one could suppose that the deleterious effect would be more pronounced. To protect the cells, I would extend the fixation time and look at fixing before preparing the agar pellet. Or use the thrombin method instead (ask your Haematology department for expired thromboplastin from their clotting tests, use expired plasma from blood bank, add a few drops of 1% calcium chloride and a clot should form in 20seconds or so. Add NBF, fix for longer than an hour (4hr to overnight is better. And process as usual. Since the clots tend to be only about 3mm in greatest dimension (depending on the volume of thromboplastin and plasma used, a short gentle program will suffice (5 changes of alcohol, 20min each: 2 changes of xylene 30min each, 3 changes of wax, 30 min each). Alternatively you could mix the pellet with some egg albumin, add an equal volume of 10% formalin in alcohol, a nice clot will form, fix for a few hours and process from alcohol (Based on the paper, with modifications, by Yamamoto et al (1985) Am J Clin Pathol 83:409-414). Interstitial glycosaminoglycans and other mucins are also water soluble so fixing in formal alcohol should retain more of these mucins than aqueous NBF fixation (Nathan, van Deth (1983) Pathology 15:301-4) Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA From: Victor Wong [mailto:vhlw...@yahoo.com] Sent: Monday, 18 February 2013 6:36 PM To: Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Dear Tony, Thank you for your prompt reply and the paper. I do fix the cell before processing in agarose and after embedding in agarose. It is inevitable to process in wax at higher than 45C as it is set by the routine workers. It is the first time I worked on cell block precessing. Do you have protocol of processing chondrogenic pellet by tissue processor? Any adjustment to my procesing and staining protocol? I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed. Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat? Best Regards, Victor From: Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au To: 'Victor Wong' vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:45 AM Subject: RE: [Histonet] Safranin O staining Victor, Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136). I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong Sent: Monday, 18 February 2013 1:34 PM To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I
RE: [Histonet] Safranin O staining
I was just thinking about Russ Allison the other day. He is certainly missed. Jean Mitchell, BS HT (ASCP) University of Wisconsin Hospital Clinics Neuromuscular Laboratory 600 Highland Avenue Madison, WI 53792-5132 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tony Henwood (SCHN) Sent: Monday, February 18, 2013 4:44 PM To: 'Victor Wong'; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Safranin O staining Hi Victor, I suppose the take home message from Russ's paper (God rest his soul - we do miss him) is that heat will affect some stains so extrapolating to routine processing temperatures, one could suppose that the deleterious effect would be more pronounced. To protect the cells, I would extend the fixation time and look at fixing before preparing the agar pellet. Or use the thrombin method instead (ask your Haematology department for expired thromboplastin from their clotting tests, use expired plasma from blood bank, add a few drops of 1% calcium chloride and a clot should form in 20seconds or so. Add NBF, fix for longer than an hour (4hr to overnight is better. And process as usual. Since the clots tend to be only about 3mm in greatest dimension (depending on the volume of thromboplastin and plasma used, a short gentle program will suffice (5 changes of alcohol, 20min each: 2 changes of xylene 30min each, 3 changes of wax, 30 min each). Alternatively you could mix the pellet with some egg albumin, add an equal volume of 10% formalin in alcohol, a nice clot will form, fix for a few hours and process from alcohol (Based on the paper, with modifications, by Yamamoto et al (1985) Am J Clin Pathol 83:409-414). Interstitial glycosaminoglycans and other mucins are also water soluble so fixing in formal alcohol should retain more of these mucins than aqueous NBF fixation (Nathan, van Deth (1983) Pathology 15:301-4) Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA From: Victor Wong [mailto:vhlw...@yahoo.com] Sent: Monday, 18 February 2013 6:36 PM To: Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Dear Tony, Thank you for your prompt reply and the paper. I do fix the cell before processing in agarose and after embedding in agarose. It is inevitable to process in wax at higher than 45C as it is set by the routine workers. It is the first time I worked on cell block precessing. Do you have protocol of processing chondrogenic pellet by tissue processor? Any adjustment to my procesing and staining protocol? I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed. Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat? Best Regards, Victor From: Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au To: 'Victor Wong' vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:45 AM Subject: RE: [Histonet] Safranin O staining Victor, Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136). I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong Sent: Monday, 18 February 2013 1:34 PM To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt
Re: [Histonet] Safranin O staining
Hi Ray, Thank you for your suggestion and the paper. There is a wonderful staining with safranin O compared to our staining that was very faint, nearly as unstained with safranin O (there was staining with fast green and hx). I dealed with sample as pellet in Falcon tubes, after experiment by my colleaque. As far as I know, we isolated mesenchymal stem cells from marrow and cultivated in chondrogenic medium for days. We don't have a pellet positive control but including a section with cartilage is useful. I really concerned that high temperature during melting the 2% agarose may be deletrious to the staining. As suggested, I will extend the fixation time. Best Regards, Victor From: koelli...@comcast.net koelli...@comcast.net To: Victor Wong vhlw...@yahoo.com Cc: histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 10:42 PM Subject: Re: [Histonet] Safranin O staining Victor, I completely agree with Tony and Renee's methodology assessment but I would also ask you to look at your induced chondrogenesis model (I've never done this but have done many, many other cell culture models). If you go to this paper http://biotech.korea.ac.kr/bk21/bbs/upload/bk21bbs_cur_165_0.pdf in Tissue Engineering they describe in detail exactly what you are trying to do, if I read your question correctly. Their pictures of safrinin 0 staining of pelleted cells from culture could be described as light staining if you compare them to the red/orange color what we all know safranin o looks like on articular surfaces. I think that is a POOR positive control for their experiments but that's just me. In-vitro cultured cells prepared for histology will seldom stain the same as in-vivo material prepared for histology. And they had to hit cells pretty hard, 5ng TGF to see some staining. Have no idea what your cell culture methodologies are but what I think you are doing is all in that paper and you can see the results in the Safranin o pictures. There are ways to get better, more similar, positive staining controls then they used. Ray Koelling Research Scientist University of Washington Seattle From: Victor Wong vhlw...@yahoo.com To: histonet@lists.utsouthwestern.edu Sent: Sunday, February 17, 2013 6:34:12 PM Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O staining
Hi Rene, Thanks for your great suggestion. Yes, I'll extend the fixation time. Do you think the embedding with melting agarose will be deletrious to the safranin staining? I just use a heating water bath to melt the 2% agarose without temperature control. I checked for complete melting and it was not boiled actually. Can I go through routine automatic tissue processing instead of manually? For Weigert staining, we had a strong nuclear staining compared with faint or unstained red colour. Any enlightenment is welcomed. Best Regards, Victor From: Rene J Buesa rjbu...@yahoo.com To: Victor Wong vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 8:42 PM Subject: Re: [Histonet] Safranin O staining Believe it or not, not because you are dealing with cells (small as they are) they require longer than 1 hour to be correctly fixed. This is what I would do: fix the cells for 8 hours minimum → prepare the pellet in agarose → process to paraffin (FFPE) block → section as usual. Now, check the distaining protocol, consult any histotechnique book because I think 2 min is Weigert is a very short time (remember the mordant) → try different staining times, or follow a known protocol (you can find that in either Peter Gray's or Lillie's books). René J. From: Victor Wong vhlw...@yahoo.com To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Sunday, February 17, 2013 9:34 PM Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O staining
Hi Tony, I'll extend the fixation, also as suggested by Rene. And then proceed to manual process. Is automatic tissue processing fine with these samples? It is interesting to process with albumin. Both of the paper cannot be accessed in our lab and I'll try to find from other sources. P.S. unforuntuately we don't have a Haematology department but may be we can try other cells. Best Regards, Victor From: Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au To: 'Victor Wong' vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Tuesday, February 19, 2013 6:44 AM Subject: RE: [Histonet] Safranin O staining Hi Victor, I suppose the take home message from Russ’s paper (God rest his soul – we do miss him) is that heat will affect some stains so extrapolating to routine processing temperatures, one could suppose that the deleterious effect would be more pronounced. To protect the cells, I would extend the fixation time and look at fixing before preparing the agar pellet. Or use the thrombin method instead (ask your Haematology department for expired thromboplastin from their clotting tests, use expired plasma from blood bank, add a few drops of 1% calcium chloride and a clot should form in 20seconds or so. Add NBF, fix for longer than an hour (4hr to overnight is better. And process as usual. Since the clots tend to be only about 3mm in greatest dimension (depending on the volume of thromboplastin and plasma used, a short gentle program will suffice (5 changes of alcohol, 20min each: 2 changes of xylene 30min each, 3 changes of wax, 30 min each). Alternatively you could mix the pellet with some egg albumin, add an equal volume of 10% formalin in alcohol, a nice clot will form, fix for a few hours and process from alcohol (Based on the paper, with modifications, by Yamamoto et al (1985) Am J Clin Pathol 83:409-414). Interstitial glycosaminoglycans and other mucins are also water soluble so fixing in formal alcohol should retain more of these “mucins” than aqueous NBF fixation (Nathan, van Deth (1983) Pathology 15:301-4) Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 thechildren'shospitalat westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA From:Victor Wong [mailto:vhlw...@yahoo.com] Sent: Monday, 18 February 2013 6:36 PM To: Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Dear Tony, Thank you for your prompt reply and the paper. I do fix the cell before processing in agarose and after embedding in agarose. It is inevitable to process in wax at higher than 45C as it is set by the routine workers. It is the first time I worked on cell block precessing. Do you have protocol of processing chondrogenic pellet by tissue processor? Any adjustment to my procesing and staining protocol? I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed. Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat? Best Regards, Victor From:Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au To: 'Victor Wong' vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:45 AM Subject: RE: [Histonet] Safranin O staining Victor, Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136). I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong Sent: Monday, 18 February 2013 1:34 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom
Re: [Histonet] Safranin O staining
Hi Liz, Thank you for your response and your invaluable experience in processing such samples. I'll try manual processing on next batch of samples. I am new to handle these samples. There would be a lot of cell debris during culture mixing with small pellet and this may be much bigger than the pellet. May I know how to get rid of it? Someone suggested to stain the pellet with eosin and place it in folded Kimwipe for processing. Is routine automatic staining helpful as we don't have a oven to melt paraffin nearby? We need to take a 30-minute walk to the lab for processing. BTW, I don't have access right to the paper. Anyway, the staining pictures on the website are very nice. Once, thank you for your help. Best Regards, Victor From: Elizabeth Chlipala l...@premierlab.com To: Victor Wong vhlw...@yahoo.com; Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:37 PM Subject: RE: [Histonet] Safranin O staining We have processed and stained these types of samples before. We do not embed the pellets in agarose prior to processing. We dye them with eosin and place them in a tea bag and process on a very short cycle - 10 minutes per station, embed section and stain. We have stained with safranin O, alcian blue, toluidine blue and IHC for collagen II. The safranin O comes out just fine as well as the others. We just receive the samples so I do not know anything about the preparation, collection and fixation. It possibly could be your construct, we have processed hundreds of these samples and the staining intensity and patterns do change. I expect this is due to the type of preparation. If you want to see pictures of the staining, you can go to this website: http://bioreagents.lifemapsc.com/products/purestem-chondropkg-4d20-8 or this paper: Sternberg H, Murai JT, Erickson IE, Funk WD, Das S, Wang Q, Snyder E, Chapman KB, Vangsness CT Jr, West MD. 2012 A human embryonic stem cell-derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme. Regen Med. 7(4):481-501 Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong [vhlw...@yahoo.com] Sent: Monday, February 18, 2013 12:35 AM To: Tony Henwood (SCHN); histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Safranin O staining Dear Tony, Thank you for your prompt reply and the paper. I do fix the cell before processing in agarose and after embedding in agarose. It is inevitable to process in wax at higher than 45C as it is set by the routine workers. It is the first time I worked on cell block precessing. Do you have protocol of processing chondrogenic pellet by tissue processor? Any adjustment to my procesing and staining protocol? I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed. Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat? Best Regards, Victor From: Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au To: 'Victor Wong' vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:45 AM Subject: RE: [Histonet] Safranin O staining Victor, Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136). I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong Sent: Monday, 18 February 2013 1:34 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis
[Histonet] Safranin O staining
Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O staining
Hi Victor I have been using agar cell blocks for over 30 years. It has been my experience that it is better to fix the cells in formalin prior to embedding in the agar to prevent damage to the cells from the heat of the agar. Another important step is to gently heat the agar so that it is just above melting point to also minimise the heat affect. If you are using microwaves to melt your agar do the following. -calibrate your microwave so that the temperature achieved is minimal -do not set and walk away, watch and stop heating as soon as melting is achieved -aliquot agar into small batches as the microwaves affect the agar such that the melting point increases with each use elevating the temperature of the agar. regards Tony Tony Reilly B.App.Sc. , M.Sc. Chief Scientist, Anatomical Pathology Pathology Queensland-PA Laboratory Health Services Support Agency | Department of Health Level 1, Building 15,Princess Alexandra Hospital Ipswich Road,WOOLLOONGABBA Qld4102 Ph: 07 3176 2412 Mob: 0402 139411 Fax: 07 3176 2930 Email: tony_rei...@health.qld.gov.au Web: www.health.qld.gov.au/qhcss/ Victor Wong vhlw...@yahoo.com 2/18/2013 12:34 pm Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This email, including any attachments sent with it, is confidential and for the sole use of the intended recipient(s). This confidentiality is not waived or lost, if you receive it and you are not the intended recipient(s), or if it is transmitted/received in error. Any unauthorised use, alteration, disclosure, distribution or review of this email is strictly prohibited. The information contained in this email, including any attachment sent with it, may be subject to a statutory duty of confidentiality if it relates to health service matters. If you are not the intended recipient(s), or if you have received this email in error, you are asked to immediately notify the sender by telephone collect on Australia +61 1800 198 175 or by return email. You should also delete this email, and any copies, from your computer system network and destroy any hard copies produced. If not an intended recipient of this email, you must not copy, distribute or take any action(s) that relies on it; any form of disclosure, modification, distribution and/or publication of this email is also prohibited. Although Queensland Health takes all reasonable steps to ensure this email does not contain malicious software, Queensland Health does not accept responsibility for the consequences if any person's computer inadvertently suffers any disruption to services, loss of information, harm or is infected with a virus, other malicious computer programme or code that may occur as a consequence of receiving this email. Unless stated otherwise, this email represents only the views of the sender and not the views of the Queensland Government. ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O staining
Dear Tony, Thanks for your prompt reply. I fix the cells in 10% NBF for 1 hour, then changed to PBS and keep at 4C fridge. To prepare cell block, I use a heating water bath and I usually need to set the heater to higher than 150C to liquidify the agarose (2% in PBS) without boiling. The agar did not melt when heated at lower temperature. I do think the agar is at less than hundred degree C but may be at 70C. After then, I trimmed the block and fix again in formalin before putting in 70% alcohol. Any comments? This is the first time I am working on cell block. May I have your processing protocol? Best Regards, Victor From: Tony Reilly tony_rei...@health.qld.gov.au To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu; Victor Wong vhlw...@yahoo.com Sent: Monday, February 18, 2013 1:50 PM Subject: Re: [Histonet] Safranin O staining Hi Victor I have been using agar cell blocks for over 30 years. It has been my experience that it is better to fix the cells in formalin prior to embedding in the agar to prevent damage to the cells from the heat of the agar. Another important step is to gently heat the agar so that it is just above melting point to also minimise the heat affect. If you are using microwaves to melt your agar do the following. -calibrate your microwave so that the temperature achieved is minimal -do not set and walk away, watch and stop heating as soon as melting is achieved -aliquot agar into small batches as the microwaves affect the agar such that the melting point increases with each use elevating the temperature of the agar. regards Tony Tony Reilly B.App.Sc. , M.Sc. Chief Scientist, Anatomical Pathology Pathology Queensland-PA Laboratory Health Services Support Agency | Department of HealthLevel 1, Building 15,Princess Alexandra Hospital Ipswich Road,WOOLLOONGABBA Qld4102 Ph: 07 3176 2412 Mob: 0402 139411 Fax: 07 3176 2930 Email: tony_rei...@health.qld.gov.au Web: www.health.qld.gov.au/qhcss/ Victor Wong vhlw...@yahoo.com 2/18/2013 12:34 pm Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This email, including any attachments sent with it, is confidential and for the sole use of the intended recipient(s). This confidentiality is not waived or lost, if you receive it and you are not the intended recipient(s), or if it is transmitted/received in error. Any unauthorised use, alteration, disclosure, distribution or review of this email is strictly prohibited. The information contained in this email, including any attachment sent with it, may be subject to a statutory duty of confidentiality if it relates to health service matters. If you are not the intended recipient(s), or if you have received this email in error, you are asked to immediately notify the sender by telephone collect on Australia +61 1800 198 175 or by return email. You should also delete this email, and any copies, from your computer system network and destroy any hard copies produced. If not an intended recipient of this email, you must not copy, distribute or take any action(s) that relies on it; any form of disclosure, modification, distribution and/or publication of this email is also prohibited. Although Queensland Health takes all reasonable steps to ensure this email does not contain malicious software, Queensland Health does not accept responsibility for the consequences if any person's computer inadvertently suffers any disruption to services, loss of information, harm or is infected with a virus, other malicious computer programme or code that may occur as a consequence of receiving this email. Unless
Re: [Histonet] Safranin O staining
Dear Tony, Thank you for your prompt reply and the paper. I do fix the cell before processing in agarose and after embedding in agarose. It is inevitable to process in wax at higher than 45C as it is set by the routine workers. It is the first time I worked on cell block precessing. Do you have protocol of processing chondrogenic pellet by tissue processor? Any adjustment to my procesing and staining protocol? I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed. Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat? Best Regards, Victor From: Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au To: 'Victor Wong' vhlw...@yahoo.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 18, 2013 11:45 AM Subject: RE: [Histonet] Safranin O staining Victor, Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136). I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Victor Wong Sent: Monday, 18 February 2013 1:34 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Safranin O
Hello everyone, I am trying to stain for cartilage in mouse skull (cross-sectioning with the brain in tact) and wanted to know if anyone might have a good protocol for Safranin O / Fast green or Safranin O / von Kossa? The tissue is embedded in MMA and was cut on a saw microtome and finished down to an approximate 20 to 30 micron tissue thickness - 50 microns including the glue layer (I glue a slide to the block before making the cut and then grind and polish to finish). I tried Weigert's hematoxylin / Safranin O / Fast Green ( I began with Weigert's Hematoxylin, followed by Fast Green and finished with Safranin O), but the Safranin O seemed to overpower the Fast Green (the calcified bone stained more red than green). Any help on this issue would be greatly appreciated as always. Thanks again. Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O
Have you tried Sanderson's Rapid Bone Stain? Try using it first (7 minutes at 60C), then rinse in dH2O (a few dip an dunks at 60C and blot dry), then counterstain with safranin O for 2-5 minutes (room temp - check intensity) and rinse in 100% EtOH (room temp - few dip and dunks and blot dry). If saf o is too intense, you should be able to differentiate a little with 95% EtOH first then do the 100% EtOH step. On a curious note, is there any particular reason why you are cutting thick sections and grinding? You should very easily be able to cut thin sections with a rotary microtome using a tungsten-carbide knife at 5 microns and then you would have greater staining flexibility. Call me if you want to discuss (317-281-1975). Jack On Feb 2, 2011, at 4:18 PM, Herrick, James L. (Jim) herrick.ja...@mayo.edu wrote: Hello everyone, I am trying to stain for cartilage in mouse skull (cross-sectioning with the brain in tact) and wanted to know if anyone might have a good protocol for Safranin O / Fast green or Safranin O / von Kossa? The tissue is embedded in MMA and was cut on a saw microtome and finished down to an approximate 20 to 30 micron tissue thickness - 50 microns including the glue layer (I glue a slide to the block before making the cut and then grind and polish to finish). I tried Weigert's hematoxylin / Safranin O / Fast Green ( I began with Weigert's Hematoxylin, followed by Fast Green and finished with Safranin O), but the Safranin O seemed to overpower the Fast Green (the calcified bone stained more red than green). Any help on this issue would be greatly appreciated as always. Thanks again. Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O stain in plastic sections
Yes, it works quite well for both demineralized and undemineralized bone sections embedded in resin. I perform MMA embedding (not a kit) and get really nice undemineralized results. The resin I use allows me the opportunity to deplastify the sections just as one would deparaffinize when working with paraffin. Jack Ratliff On Jan 30, 2009, at 8:47 AM, Markus F. Meyenhofer mi...@superlink.net wrote: Hello all. Does Safranin O staining work on plastic sections (bone, decalcified)? Any help would be appreciated. Regards, Markus ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Safranin O stain in plastic sections
Gotta second this. I've done a lot of PMMA and/or Spurrs-embedded sectioning with it with good results. Jack Ratliff wrote: Yes, it works quite well for both demineralized and undemineralized bone sections embedded in resin. I perform MMA embedding (not a kit) and get really nice undemineralized results. The resin I use allows me the opportunity to deplastify the sections just as one would deparaffinize when working with paraffin. Jack Ratliff On Jan 30, 2009, at 8:47 AM, Markus F. Meyenhofer mi...@superlink.net wrote: Hello all. Does Safranin O staining work on plastic sections (bone, decalcified)? Any help would be appreciated. Regards, Markus ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
