[Histonet] Question regarding dehydration
Hi there, we are having problems trying to cut some embedded samples (they crumble in the bath and the few cuts we manage to get into HE are crap). These are formalin fixed samples (bovine foetal and placenta samples) which went straight from formlin into 100º ethanol for the dehydration before clearing. I was wondering if such drastic dehydration step (no 60º, 70º or 90º ethanol before the 100º) could have damage the tissue. Has anyone have a similar issue before? do you think the samples are ruined for histology? Thanks a lot for your help Regards Julio Instituto de Ganaderia de Montaña Spain ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC
[Cost of the reagent / (Volume of stock reagent / volume used per slide)]. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jb Sent: Thursday, February 12, 2015 7:29 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC I am new to IHC, can anyone explain an easy way to calculate the equation of IHC cost per slide? Thank you for your help... Sincerely, Craig Sent from my iPhone ___ 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] Question regarding dehydration
Hi Ryan, thanks a lot for your thoughts. These blocks were processed elsewhere and sent to us for the cutting and staining. Tissues were dehydrated in five consecutive baths of ethanol 100%, 5 hours each (manual processing). Then, they went to xilene (three baths, 4 hours each) and paraffin (two batch, 1h30´ each). Truth is that I have never seen such processing before. In our lab, with automatic processing, we begin with 60% ethanol and go to 70, then 95 and then 100 before ethanol/xylene. I was wondering if anybody has used the 100% ethanol processing before and which was the influence over tissues. Thanks Julio Forwarded Message Subject:RE: [EXTERNAL] [Histonet] Question regarding dehydration Date: Fri, 13 Feb 2015 09:06:47 -0500 From: Roy, Ryan ryan@va.gov To: 'Julio Benavides' j.benavi...@eae.csic.es If the tissue is fatty it will blow apart on the water bath. If the H and E staining is poor its probably the processing. What is the exact procedure for your process including time that the tissue was in each reagent? I would include this in a message to all of histonet as well as some may more knowledgeable than myself. Also, I would recommend using a 95% solution prior to the 100% alcohol (s) if not a 70-80% and 95%. Ryan Roy HTL (ASCP) Manchester Veterans Affairs Medical Center Manchester New Hampshire Disclosure: The content of this email does not represent the views or opinons of the VA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio Benavides Sent: Friday, February 13, 2015 4:26 AM To: histonet@lists.utsouthwestern.edu Subject: [EXTERNAL] [Histonet] Question regarding dehydration Hi there, we are having problems trying to cut some embedded samples (they crumble in the bath and the few cuts we manage to get into HE are crap). These are formalin fixed samples (bovine foetal and placenta samples) which went straight from formlin into 100º ethanol for the dehydration before clearing. I was wondering if such drastic dehydration step (no 60º, 70º or 90º ethanol before the 100º) could have damage the tissue. Has anyone have a similar issue before? do you think the samples are ruined for histology? Thanks a lot for your help Regards Julio Instituto de Ganaderia de Montaña Spain ___ 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: [EXTERNAL] RE: [Histonet] Question regarding dehydration
Thanks Ryan. Tissu sections where thin, something about 2 and 3 mm. I think they were well fixed (buffered formalin) nut the problem was the dehydration. to go from formalin straight into 100% ethanol looks a bit too drastic to me. Thanks for your thoughts Julio On 13/02/2015 16:44, Roy, Ryan wrote: Its well documented in the literature that using graded alcohols in processing is advantageous to prevent hardening of the tissue. How thick are the tissue section cut that are being processed? It is also well documented that sections should avoid being cut thicker than 3-4mm as this prevents the penetration the fixative as well as the other reagents. 4 hours in each reagent seems excessive... ask other people too since I have limited experience. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio Benavides Sent: Friday, February 13, 2015 10:19 AM To: histonet@lists.utsouthwestern.edu Subject: [EXTERNAL] RE: [Histonet] Question regarding dehydration Hi Ryan, thanks a lot for your thoughts. These blocks were processed elsewhere and sent to us for the cutting and staining. Tissues were dehydrated in five consecutive baths of ethanol 100%, 5 hours each (manual processing). Then, they went to xilene (three baths, 4 hours each) and paraffin (two batch, 1h30´ each). Truth is that I have never seen such processing before. In our lab, with automatic processing, we begin with 60% ethanol and go to 70, then 95 and then 100 before ethanol/xylene. I was wondering if anybody has used the 100% ethanol processing before and which was the influence over tissues. Thanks Julio Forwarded Message Subject:RE: [EXTERNAL] [Histonet] Question regarding dehydration Date: Fri, 13 Feb 2015 09:06:47 -0500 From: Roy, Ryan ryan@va.gov To: 'Julio Benavides' j.benavi...@eae.csic.es If the tissue is fatty it will blow apart on the water bath. If the H and E staining is poor its probably the processing. What is the exact procedure for your process including time that the tissue was in each reagent? I would include this in a message to all of histonet as well as some may more knowledgeable than myself. Also, I would recommend using a 95% solution prior to the 100% alcohol (s) if not a 70-80% and 95%. Ryan Roy HTL (ASCP) Manchester Veterans Affairs Medical Center Manchester New Hampshire Disclosure: The content of this email does not represent the views or opinons of the VA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio Benavides Sent: Friday, February 13, 2015 4:26 AM To: histonet@lists.utsouthwestern.edu Subject: [EXTERNAL] [Histonet] Question regarding dehydration Hi there, we are having problems trying to cut some embedded samples (they crumble in the bath and the few cuts we manage to get into HE are crap). These are formalin fixed samples (bovine foetal and placenta samples) which went straight from formlin into 100º ethanol for the dehydration before clearing. I was wondering if such drastic dehydration step (no 60º, 70º or 90º ethanol before the 100º) could have damage the tissue. Has anyone have a similar issue before? do you think the samples are ruined for histology? Thanks a lot for your help Regards Julio Instituto de Ganaderia de Montaña Spain ___ 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
RE: [EXTERNAL] RE: [Histonet] Question regarding dehydration
Julio 4 to 5 hours a station is way to long for processing samples of this size, regardless of what type of tissue or species they are from. Graduated alcohol dehydration is better. I would process samples of that size (unless it was bone, fat or skin) for no longer than 45 - 60 minutes a solution for manual processing. Here is what a typical processing cycle would look like once the tissue has been adequately fixed. 50% alcohol (you want to start here if you are working with small animal tissue such as mice and rats) 70% alcohol 80% alcohol 95% alcohol 100% alcohol 100% alcohol Xylene Xylene Paraffin Paraffin Paraffin I hope this helps, you may be able to get sections by trimming into the block and then soaking them on wet ice for some time (possibly an hour or so). Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell l...@premierlab.com www.premierlab.com March 10, 2014 is Histotechnology Professionals Day Ship to Address: Premier Laboratory, LLC 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 Julio Benavides Sent: Friday, February 13, 2015 8:43 AM To: histonet@lists.utsouthwestern.edu Subject: Re: [EXTERNAL] RE: [Histonet] Question regarding dehydration Thanks Ryan. Tissu sections where thin, something about 2 and 3 mm. I think they were well fixed (buffered formalin) nut the problem was the dehydration. to go from formalin straight into 100% ethanol looks a bit too drastic to me. Thanks for your thoughts Julio On 13/02/2015 16:44, Roy, Ryan wrote: Its well documented in the literature that using graded alcohols in processing is advantageous to prevent hardening of the tissue. How thick are the tissue section cut that are being processed? It is also well documented that sections should avoid being cut thicker than 3-4mm as this prevents the penetration the fixative as well as the other reagents. 4 hours in each reagent seems excessive... ask other people too since I have limited experience. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio Benavides Sent: Friday, February 13, 2015 10:19 AM To: histonet@lists.utsouthwestern.edu Subject: [EXTERNAL] RE: [Histonet] Question regarding dehydration Hi Ryan, thanks a lot for your thoughts. These blocks were processed elsewhere and sent to us for the cutting and staining. Tissues were dehydrated in five consecutive baths of ethanol 100%, 5 hours each (manual processing). Then, they went to xilene (three baths, 4 hours each) and paraffin (two batch, 1h30´ each). Truth is that I have never seen such processing before. In our lab, with automatic processing, we begin with 60% ethanol and go to 70, then 95 and then 100 before ethanol/xylene. I was wondering if anybody has used the 100% ethanol processing before and which was the influence over tissues. Thanks Julio Forwarded Message Subject: RE: [EXTERNAL] [Histonet] Question regarding dehydration Date: Fri, 13 Feb 2015 09:06:47 -0500 From: Roy, Ryan ryan@va.gov To: 'Julio Benavides' j.benavi...@eae.csic.es If the tissue is fatty it will blow apart on the water bath. If the H and E staining is poor its probably the processing. What is the exact procedure for your process including time that the tissue was in each reagent? I would include this in a message to all of histonet as well as some may more knowledgeable than myself. Also, I would recommend using a 95% solution prior to the 100% alcohol (s) if not a 70-80% and 95%. Ryan Roy HTL (ASCP) Manchester Veterans Affairs Medical Center Manchester New Hampshire Disclosure: The content of this email does not represent the views or opinons of the VA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio Benavides Sent: Friday, February 13, 2015 4:26 AM To: histonet@lists.utsouthwestern.edu Subject: [EXTERNAL] [Histonet] Question regarding dehydration Hi there, we are having problems trying to cut some embedded samples (they crumble in the bath and the few cuts we manage to get into HE are crap). These are formalin fixed samples (bovine foetal and placenta samples) which went straight from formlin into 100º ethanol for the dehydration before clearing. I was wondering if such drastic dehydration step (no 60º, 70º or 90º ethanol before the 100º) could have damage the tissue. Has anyone have a similar issue before? do you think the samples are ruined for histology? Thanks a lot for your help Regards Julio Instituto de Ganaderia de Montaña Spain
[Histonet] Villanueva stain
Histonetters can any one of you please share the method of preparing this stain as well the staining methoďology. Thank you Rooki ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
: [Histonet] Retic Kit Problems
We use the Ventana Nexes and have the same issues with the Retic and the GMS kit. We have had multiple types of tissue and controls that would not stain (including bone marrow). Repeat stains have to be done when this is happening wasting time and reagent. We take the kit out with ample time to warm up, have tried doing more decon frequency (sometimes each week or more) and have not found a specific cause for the staining or lack of staining. Histology voodoo Classification: Internal and External Use\\Not VA Sensitive This message has been categorized by White, Lisa M. on Friday, February 13, 2015 at 2:08:44 PM in accordance with VA Handbook 6500 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Frozen tissue/OCT Vials
Hi Brian, we use Nalgene cryovials with screw top for frozen muscle. Regards Ranna ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC
need your vendor pricing for detection, ancillaries and antibody. Divide by amount applied/used per test. Add in materials costs and a labor value. Add in overhead. Joelle Weaver MAOM, HTL (ASCP) QIHC From: craiga...@gmail.com Date: Thu, 12 Feb 2015 19:29:20 -0700 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC I am new to IHC, can anyone explain an easy way to calculate the equation of IHC cost per slide? Thank you for your help... Sincerely, Craig Sent from my iPhone ___ 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] HISTOPALOOZA 2015 SECURE YOUR ROOM
Don't forget to pack your suit for the heated pool at the Lodge at Lake Lanier Islands. Bring your campfire songs and stories for s'mores by the fire pit. Personally, I have flashbacks when I think about s'mores by the campfire. I was in 4th grade on a Girl Scout camping trip and I had sand in my s'mores. I was too scared to tell my leader so I ate it anyway. I would have been in tip top shape if I had a gizzard, I suppose. Meet in the Bullfrog lounge for adult beverages or on the veranda with a mint julep. Come get your geek on at the GSH HISTOPALOOZA. We will keep the sand outta the s'mores for ya! Stay tuned for featured speakers. And, if anyone goes to see Fifty Shades drop me a line here. :) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
