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] Benchmarking information
Way to go Rene! Are you a politician on the side? LOL Just kidding - couldn't help myself! :) Yours, Dave From: Rene J Buesa rjbu...@yahoo.com To: joellewea...@hotmail.com joellewea...@hotmail.com Cc: Histonet histonet@lists.utsouthwestern.edu; sheila.tap...@essentiahealth.org sheila.tap...@essentiahealth.org Sent: Sunday, February 17, 2013 11:15 AM Subject: Re: [Histonet] Benchmarking information Joel: It is not that I did not care about your response, it is that it was not a good advise. You have to remind that when somebody asks HistoNet it is because they do not know about the question and are seeking advise and very probably they will follow the advise. If somebody tries to improve the work flow of the lab and its TAT that person cannot look inwards because they will just understand their operation and that does not guarantee improvement. The only way you have to improve is, after knowing what you do, is to compare and emulate those who have the best rates. Competition is the basis and you cannot compete if you only look inwards. Your initial advise was correct (as I state) and allowed to know yourself but the improvement process has to expand to others. I also stated that I intended no offense so, if you felt offended, please forgive me. I was only trying to help a fellow HistoNeter! René J. From: joellewea...@hotmail.com joellewea...@hotmail.com To: Rene J Buesa rjbu...@yahoo.com; sheila.tap...@essentiahealth.org sheila.tap...@essentiahealth.org; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Sunday, February 17, 2013 10:52 AM Subject: Re: [Histonet] Benchmarking information Sorry you did not care for my two cents, just trying to help Sent from my Verizon Wireless 4G LTE Smartphone - Reply message - From: Rene J Buesa rjbu...@yahoo.com To: joelle weaver joellewea...@hotmail.com, sheila.tap...@essentiahealth.org sheila.tap...@essentiahealth.org, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Subject: [Histonet] Benchmarking information Date: Sat, Feb 16, 2013 9:54 am I feel the urge of commenting on this advise about how to determine a benchmark for a histology lab. 1- I agree that you should first know your lab and how long it takes for your techs to complete the work-flow from receiving the specimens to reporting the final diagnosis BUT 2- if you just stop at that point you will be unable to determine if you have a problem. Finding problems and developing solutions can only be achieved if you COMPARE your lab to others and how efficient you are, how your TAT in each step of the process compares with with other labs of the same size (similar workload, staff and technology). 3- unless you compare you cannot develop strategies to do at least as well as others do. 4- to achieve the comparison objective you have to have information about other labs and you can only find about that if you hire a consultant (very expensive) or if you reffer yourself to published information. I think that you will find useful some articles I have written on the subject and that you can find at http://www.histosearch.com/rene.html Finally, the article you have been referred to (http://www.biomedcentral.com/1472-6890/10/2) in its original version was a propaganda for the VentanaSymphony autostainer and had to be rewritten to achieve its present form. Essentially lacks the information you need (no offense intended, just the facts from my point of view). René J. From: joelle weaver joellewea...@hotmail.com To: sheila.tap...@essentiahealth.org; histonet@lists.utsouthwestern.edu Sent: Friday, February 15, 2013 5:09 PM Subject: RE: [Histonet] Benchmarking information The best option is your own process mapping and time studies of these processes working in your own lab environment. It is usually easiest if you intially break it down into the preanalyic, analytic and post analytic phases and the critical variables that reduce your delivery times or contribute to impact each of the bottlenecks/delays/errors/ problems, in each phasse, and then put these in problem statements in your LSS project charters. It gets convoluted if you put them all in one, so I think it is best if you find one that you can target as a quick win and use the momentum from this for further projects. Do a detailed as is process map from your current data and then work on streamlining by elimination of those items to build a more standardized and improved how you want it to be process map. This becomes your SOP more or less for the new procedure. I know you wanted numbers, but I guess I feel your own numbers are going to be a lot more useful. However, there are a few references out there that are pretty detailed and are more or less case studies that have some numerical data. For example-The combined positive impact of Lean methodology and Ventana Symphony autostainer on histology lab workflow-
Re: [Histonet] Benchmarking information
No politician here, just plain old! René J. From: David Kemler histot...@yahoo.com To: Rene J Buesa rjbu...@yahoo.com; Fellow HistoNetters Histonet@Lists.UTSouthwestern.edu Sent: Monday, February 18, 2013 11:41 AM Subject: Re: [Histonet] Benchmarking information Way to go Rene! Are you a politician on the side? LOL Just kidding - couldn't help myself! :) Yours, Dave From: Rene J Buesa rjbu...@yahoo.com To: joellewea...@hotmail.com joellewea...@hotmail.com Cc: Histonet histonet@lists.utsouthwestern.edu; sheila.tap...@essentiahealth.org sheila.tap...@essentiahealth.org Sent: Sunday, February 17, 2013 11:15 AM Subject: Re: [Histonet] Benchmarking information Joel: It is not that I did not care about your response, it is that it was not a good advise. You have to remind that when somebody asks HistoNet it is because they do not know about the question and are seeking advise and very probably they will follow the advise. If somebody tries to improve the work flow of the lab and its TAT that person cannot look inwards because they will just understand their operation and that does not guarantee improvement. The only way you have to improve is, after knowing what you do, is to compare and emulate those who have the best rates. Competition is the basis and you cannot compete if you only look inwards. Your initial advise was correct (as I state) and allowed to know yourself but the improvement process has to expand to others. I also stated that I intended no offense so, if you felt offended, please forgive me. I was only trying to help a fellow HistoNeter! René J. From: joellewea...@hotmail.com joellewea...@hotmail.com To: Rene J Buesa rjbu...@yahoo.com; sheila.tap...@essentiahealth.org sheila.tap...@essentiahealth.org; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Sunday, February 17, 2013 10:52 AM Subject: Re: [Histonet] Benchmarking information Sorry you did not care for my two cents, just trying to help Sent from my Verizon Wireless 4G LTE Smartphone - Reply message - From: Rene J Buesa rjbu...@yahoo.com To: joelle weaver joellewea...@hotmail.com, sheila.tap...@essentiahealth.org sheila.tap...@essentiahealth.org, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Subject: [Histonet] Benchmarking information Date: Sat, Feb 16, 2013 9:54 am I feel the urge of commenting on this advise about how to determine a benchmark for a histology lab. 1- I agree that you should first know your lab and how long it takes for your techs to complete the work-flow from receiving the specimens to reporting the final diagnosis BUT 2- if you just stop at that point you will be unable to determine if you have a problem. Finding problems and developing solutions can only be achieved if you COMPARE your lab to others and how efficient you are, how your TAT in each step of the process compares with with other labs of the same size (similar workload, staff and technology). 3- unless you compare you cannot develop strategies to do at least as well as others do. 4- to achieve the comparison objective you have to have information about other labs and you can only find about that if you hire a consultant (very expensive) or if you reffer yourself to published information. I think that you will find useful some articles I have written on the subject and that you can find at http://www.histosearch.com/rene.html Finally, the article you have been referred to (http://www.biomedcentral.com/1472-6890/10/2) in its original version was a propaganda for the VentanaSymphony autostainer and had to be rewritten to achieve its present form. Essentially lacks the information you need (no offense intended, just the facts from my point of view). René J. From: joelle weaver joellewea...@hotmail.com To: sheila.tap...@essentiahealth.org; histonet@lists.utsouthwestern.edu Sent: Friday, February 15, 2013 5:09 PM Subject: RE: [Histonet] Benchmarking information The best option is your own process mapping and time studies of these processes working in your own lab environment. It is usually easiest if you intially break it down into the preanalyic, analytic and post analytic phases and the critical variables that reduce your delivery times or contribute to impact each of the bottlenecks/delays/errors/ problems, in each phasse, and then put these in problem statements in your LSS project charters. It gets convoluted if you put them all in one, so I think it is best if you find one that you can target as a quick win and use the momentum from this for further projects. Do a detailed as is process map from your current data and then work on streamlining by elimination of those items to build a more standardized and improved how you want it to be process map. This becomes your SOP more or less for the new procedure. I know you wanted numbers, but I guess I feel your own numbers are going to be a lot more useful. However, there are a few
[Histonet] slide and block discards
Hello All- Does anyone have some authoritative document stating that histologic slides and/or blocks are non-infectious? We all know this to be true--but is there a piece of paper that states this unequivocally? Thanks! Cheryl Kerry, HT(ASCP) Full Staff Inc. Staffing the AP Lab by helping one GREAT Tech at a time. 281.852.9457 Office 800.756.3309 Phone Fax ad...@fullstaff.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] slide and block discards
Just found this off the OSHA Website http://www.osha.gov/SLTC/bloodbornepathogens/recognition.html It lists how OSHA defines blood and it also lists the OPMI - other potentially infectious materials (unfixed tissue or organ) plus it has a bunch of guidance links on the topic. Hazard Recognition The CDC estimates that 5.6 million workers in the health care industry and related occupations are at risk of occupational exposure to bloodborne pathogens, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and others. All occupational exposure to blood or other potentially infectious materials (OPIM) place workers at risk for infection with bloodborne pathogens. OSHA defines blood to mean human blood, human blood components, and products made from human blood. Other potentially infectious materials (OPIM) means: (1) The following human body fluids: semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, any body fluid that is visibly contaminated with blood, and all body fluids in situations where it is difficult or impossible to differentiate between body fluids; (2) Any unfixed tissue or organ (other than intact skin) from a human (living or dead); and (3) HIV-containing cell or tissue cultures, organ cultures, and HIV- or HBV-containing culture medium or other solutions; and blood, organs, or other tissues from experimental animals infected with HIV or HBV. The following references aid in recognizing workplace hazards associated with bloodborne pathogens. 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 Cheryl [tkngfl...@yahoo.com] Sent: Monday, February 18, 2013 11:25 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] slide and block discards Hello All- Does anyone have some authoritative document stating that histologic slides and/or blocks are non-infectious? We all know this to be true--but is there a piece of paper that states this unequivocally? Thanks! Cheryl Kerry, HT(ASCP) Full Staff Inc. Staffing the AP Lab by helping one GREAT Tech at a time. 281.852.9457 Office 800.756.3309 Phone Fax ad...@fullstaff.org ___ 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] slide and block discards
I'd be interested in it too. Claire From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Cheryl [tkngfl...@yahoo.com] Sent: Monday, February 18, 2013 12:25 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] slide and block discards Hello All- Does anyone have some authoritative document stating that histologic slides and/or blocks are non-infectious? We all know this to be true--but is there a piece of paper that states this unequivocally? Thanks! Cheryl Kerry, HT(ASCP) Full Staff Inc. Staffing the AP Lab by helping one GREAT Tech at a time. 281.852.9457 Office 800.756.3309 Phone Fax ad...@fullstaff.org ___ 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] slides and blocks discards
I found this on the CDC website: http://www.cdc.gov/od/eaipp/faq.htm What type of material does NOT require an Etiologic Agent Import Permit? Non-infectious materials – e.g., formalin-fixed specimens, tissues, or slides, or non-infectious human stem cells or non-infectious human organs for transplantation. 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Fw: RE: [Histonet] slide and block discards
Thanks to Liz for this CDC reference!
--- On Mon, 2/18/13, Elizabeth Chlipala l...@premierlab.com wrote:
From: Elizabeth Chlipala l...@premierlab.com
Subject: RE: [Histonet] slide and block discards
To: Cheryl tkngfl...@yahoo.com
Date: Monday, February 18, 2013, 10:40 AM
#yiv640601932 P {
MARGIN-TOP:0px;MARGIN-BOTTOM:0px;}
This is from the CDC
http://www.cdc.gov/od/eaipp/faq.htm
What type of material does NOT require an Etiologic Agent Import Permit?
Non-infectious materials – e.g., formalin-fixed specimens, tissues, or slides,
or non-infectious human stem cells or non-infectious human organs for
transplantation.
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: Cheryl [tkngfl...@yahoo.com]
Sent: Monday, February 18, 2013 11:38 AM
To: Elizabeth Chlipala
Subject: RE: [Histonet] slide and block discards
Nice. So they clearly state UNFIXED but leave out FIXED tissue--which allows
the assumption that fixed tissue is not on the list thus not biohazardous.
Anything you've found that STATES this outright?
I really want to close this loop!!
Thank you!
Cheryl Kerry, HT(ASCP)
Full Staff Inc.
Staffing the AP Lab by helping one GREAT Tech at a time.
281.852.9457 Office
800.756.3309 Phone Fax
ad...@fullstaff.org
Sign up for the FREE newsletter AP News--updates, tricks of the trade and
current issues for Anatomic Pathology Clinical Labs. Send a 'subscribe' request
to apn...@fullstaff.org. Please include your name and specialty in the body of
the email.
--- On Mon, 2/18/13, Elizabeth Chlipala l...@premierlab.com wrote:
From: Elizabeth Chlipala l...@premierlab.com
Subject: RE: [Histonet] slide and block discards
To: Cheryl tkngfl...@yahoo.com, histonet@lists.utsouthwestern.edu
histonet@lists.utsouthwestern.edu
Date: Monday, February 18, 2013, 10:32 AM
Just found this off the OSHA Website
http://www.osha.gov/SLTC/bloodbornepathogens/recognition.html
It lists how OSHA defines blood and it also lists the OPMI - other potentially
infectious materials (unfixed tissue or organ) plus it has a bunch of guidance
links on the topic.
Hazard Recognition
The CDC estimates that 5.6 million workers in the health care industry and
related occupations are at risk of occupational exposure to bloodborne
pathogens, including human immunodeficiency virus (HIV), hepatitis B virus
(HBV), hepatitis C virus (HCV), and others. All occupational exposure to blood
or other potentially infectious materials (OPIM) place workers at risk for
infection with bloodborne pathogens. OSHA defines blood to mean human blood,
human blood components, and products made from human blood. Other potentially
infectious materials (OPIM) means: (1) The following human body fluids: semen,
vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid,
pericardial fluid, peritoneal fluid, amniotic fluid, saliva in dental
procedures, any body fluid that is visibly contaminated with blood, and all
body fluids in situations where it is difficult or impossible to differentiate
between body fluids; (2) Any unfixed tissue or organ (other
than intact skin) from a human (living or dead); and (3) HIV-containing cell
or tissue cultures, organ cultures, and HIV- or HBV-containing culture medium
or other solutions; and blood, organs, or other tissues from experimental
animals infected with HIV or HBV. The following references aid in recognizing
workplace hazards associated with bloodborne pathogens.
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 Cheryl
[tkngfl...@yahoo.com]
Sent: Monday, February 18, 2013 11:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] slide and block discards
Hello All-
Does anyone have some authoritative document stating that histologic slides
and/or blocks are non-infectious? We all know this to be true--but is there a
piece of paper that states this unequivocally?
Thanks!
Cheryl Kerry, HT(ASCP)
Full Staff Inc.
Staffing the AP Lab by helping one GREAT Tech at a time.
281.852.9457 Office
800.756.3309 Phone Fax
ad...@fullstaff.org
___
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[Histonet] RE: slides and blocks discards
Correct- we had to learn all this for IATA training (how to package and properly ship biological samples). There's a massive list. Bernice Bernice Frederick HTL (ASCP) Senior Research Tech Pathology Core Facility ECOGPCO-RL Robert. H. Lurie Cancer Center Northwestern University 710 N Fairbanks Court Olson 8-421 Chicago,IL 60611 312-503-3723 b-freder...@northwestern.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Elizabeth Chlipala Sent: Monday, February 18, 2013 12:45 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] slides and blocks discards I found this on the CDC website: http://www.cdc.gov/od/eaipp/faq.htm What type of material does NOT require an Etiologic Agent Import Permit? Non-infectious materials - e.g., formalin-fixed specimens, tissues, or slides, or non-infectious human stem cells or non-infectious human organs for transplantation. 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 ___ 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 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 of
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 in
