Re: [Histonet] granuloma and lymphocytes with special stains
Giemsa is the adequate protocol but perhaps your protocol is not good enough. Under separate cover I am sending mine. René J. From: Hans B Snyder h...@histologistics.com To: Histonet@lists.utsouthwestern.edu Sent: Tuesday, February 19, 2013 10:57 AM Subject: [Histonet] granuloma and lymphocytes with special stains Hello, Does anyone have a good special stain protocol for lymphocytes and or granulocytes? I have been trying PAS, Giemsa and T-Blue but the results are not specific enough. The tissue is rat hearts from 3 years ago so the antigenicy is all but gone for IHC. Thank you Hans B Snyder Histologistics 60 Prescott Street Worcester, MA 01605 508-308-7800 h...@histologistics.com ha...@histologistics.com ___ 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 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 at
[Histonet] Help!
I need to speak with someone who has experience with doing (and interpretating) in situ hybridization for EBER. Please contact me directly. Thank you. Richard Richard W. Cartun, MS, PhD Director, Histology Immunopathology Director, Biospecimen Collection Programs Assistant Director, Anatomic Pathology Hartford Hospital 80 Seymour Street Hartford, CT 06102 (860) 545-1596 (860) 545-2204 Fax ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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
