Re: [Histonet] PFA Fixed tissue not sticking to slides?
Hello Jennifer, yes over-fixation will cause adhesion problems. This applies to formalin fixed frozens as well as PFA fixed frozens. I have had good luck using chromated gelatin to coat slides. I have successfully cut and stained lung specimens left in NBF for several months. Commercially, chromated gelatin is sold as STA-ON. I believe Leica now owns it, but you can make your own quite easily. I tend to use STA-ON full strength, or minimally diluted. Spread some on a slide with a transfer pipette, taking care not to allow it to run onto the back of the slide. Shake off the excess and set vertical to dry. The coating MUST be dry before you use the slide to pick up sections. Freshly made slides work best. After pick up, it is important to air-dry the section completely before immersing it in any solution. The moisture content of the frozen section must evaporate or the chromated gelatin will not hold the tissue. Don't worry, fixed sections do not suffer from artifacts when air-dried because the morphology is already locked by the fixation. Best luck! -brice The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] PFA Fixed tissue not sticking to slides?
No...never add sucrose to "PFA" when fixing tissues for freezingor any other time. Fix, then sucrose- immerse until tissue sinks to bottom of receptacle if you can't snap-freeze effectively Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] PFA Fixed tissue not sticking to slides?
Good charged slides or subbed slides will work. In regards to the fixation, I personally find the morphology with 4% PFA O/N followed by 30% sucrose better than doing them simultaneously. Michelle ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] PFA Fixed tissue not sticking to slides?
Hi Jenny. Can you use Pwax embedding as an alternative? H&E will always be better on Pwax sections. IHC will also be in many instances. What abs are you probing with? Single/double-labelling? Yesyou are fixing for too long ( for frozen sectioningfor Pwax you can fix your specimens for a week and still not suffer IHC-problems: after HIER they will or will never work, depending upon ab) PFA( Formalin) fixed sections are well-known for not being as adherent as unfixed frozen material. Why not freeze unfixed? No sucrose required ( all that does is allow for slow freezing without, in most cases, formation of ice-crystals: good snap freezing does not need sucrose pretreatment) If you are after good morphology then Pwax is the way to go, imho. If you have to cut PFA-fixed then frozen tissues, after mounting on slides stick them in a 50C oven for 2hrs. Use Superfrost Plus slides. Then use or, store at 4C for a week or freeze. For the latter 2, ALWAYS take out of fridge/freezer and immediately place under a fan/operating fume hood. Do not allow condensation to form. Orplace them immediately into that same 50C oven for an hour. Do not do this to unfixed frozen sections! Sure, check out EC's excellent advice ( as always) re slides. Interestedly, Carl Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] PFA Fixed Tissue not sticking to slides?
Thank you Charles Scouton for responding. The slides are positive charged slides from IMEB. Thank you Elizabeth Chilpala for responding. The treatment after sectioning is one I’ve forwarded to the service that is processing for us. I only do the PFA/sucrose/embedding part. My understanding is it is pretty standard (?) for cryo sections for H&E and IHC, but I am checking that out. Jennifer Eveleth Original message below: Dear HistoFolks, Has anyone had experience with cryo-sectioned tissue that is PFA fixed not adhering to slides? The tissue is rabbit cornea culture, 4% PFA fixed O/N, then run through 20% sucrose gradient, then frozen in OCT. We think that the PFA fix is much longer than needed for a tissue this thin, and were going to try 2 hr fix. Would over-fixation cause the section to not adhere? The PFA/Sucrose fixation really improves the morphology and the IHC stains that we have run so far. Thank you in advance for your help. Jennifer Eveleth ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] PFA Fixed tissue not sticking to slides?
Are you using a good plus slide? We have started using these Tru-Bond slides when we have tissue adherence problems, they seem to work really well. How are you handling the slides once you section? 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 Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: jenny sandy via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Friday, July 29, 2016 1:48 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] PFA Fixed tissue not sticking to slides? Dear HistoFolks, Has anyone had experience with cryo-sectioned tissue that is PFA fixed not adhering to slides? The tissue is rabbit cornea culture, 4% PFA fixed O/N, then run through 20% sucrose gradient, then frozen in OCT. We think that the PFA fix is much longer than needed for a tissue this thin, and were going to try 2 hr fix. Would over-fixation cause the section to not adhere? The PFA/Sucrose fixation really improves the morphology and the IHC stains that we have run so far. Thank you in advance for your help. Jennifer Eveleth ___ 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] PFA Fixed tissue not sticking to slides?
Dear HistoFolks, Has anyone had experience with cryo-sectioned tissue that is PFA fixed not adhering to slides? The tissue is rabbit cornea culture, 4% PFA fixed O/N, then run through 20% sucrose gradient, then frozen in OCT. We think that the PFA fix is much longer than needed for a tissue this thin, and were going to try 2 hr fix. Would over-fixation cause the section to not adhere? The PFA/Sucrose fixation really improves the morphology and the IHC stains that we have run so far. Thank you in advance for your help. Jennifer Eveleth ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
