Re: [Histonet] p2y12 cryo rat problem high background
Hi Esther, I worked extensively with brain and spinal cord sections in the past, in multiple species. You need endogenous quenching step. I would always make my own (commerically available components yielded different results). I used a 0.09% H2O2 in 6% Triton-X 100: (formula: 200mL 1x PBS + 6mL H2O2 + 0.33mL 6% Triton-X 100 solution). Are you samples fixed frozen or fresh frozen? If fixed-frozen, there is no need for the acetone step. In fact, acetone in brain and spinal cord sections makes the tissue brittle. I would recommend this is skipped. If your samples are fresh-frozen, I would recommend a 95% EtOH fix. This is significantly gentler on the tissue. I have no experience with the Envision system, but the polymer system I would always use was from Cell Signaling Technologies. What antibody are you using? On Thu, Nov 15, 2018 at 7:48 AM Kooijman, E.J.M. (Esther) via Histonet < histonet@lists.utsouthwestern.edu> wrote: > Hello Bobbie, > > But should I elimination endogenous peroxidase activity in brain/spinal > cord tissues? > Brains and spinal cord was harvested after cervical dislocation, then the > tissue was snap frozen (isopentane..). > > thanks for your help, > Esther > > -Oorspronkelijk bericht- > Van: Boyce, Bobbie [mailto:bobbie.bo...@nemours.org] > Verzonden: donderdag 15 november 2018 12:21 > Aan: Kooijman, E.J.M. (Esther) > Onderwerp: RE: p2y12 cryo rat problem high background > > Hi Ester, > Try Peroxoblock (Zymed) before your BSA block, but you have to be careful > not to leave it on too long or it will eat your tissue. It's been a while > since I've had to used it. > > > Bobbie Boyce > Histology Specialist III > DuPont Experimental Station > Nemours- Biomedical Research Department > Histochemistry and Tissue Processing Core > 200 Powder Mill Road, Bldg.400 Rm.5240 > Wilmington, DE 19803 > > (lab) 302-651-6771 > (fax) 302-651-5010 > > > > -Original Message- > From: Kooijman, E.J.M. (Esther) via Histonet < > histonet@lists.utsouthwestern.edu> > Sent: Thursday, November 15, 2018 5:53 AM > To: histonet@lists.utsouthwestern.edu > Subject: [Histonet] p2y12 cryo rat problem high background > > **This is an External Email - Please DO NOT open attachments or click > links from unknown senders or unexpected email. ** > > Hello all, > > > > I am trying to stain cryo brain sections from the rat 7um but having a lot > of background. What am I doing wrong. Below the protocol is used and tried > to adjust… > > > > 1- Fix the tissue with cold acetone (-20 ⁰C) for 10 minutes > > > > 2- Let the slide dry for 30 minutes at room temperature. Isolate the > sections with DAKO pen > > > > 3- Block sections with BSA 2% in PBS for 1 hour at room temperature > > > > 4- Add primary antibody in PBS/BSA 1%, overnight at 4⁰C or 1h at room > temperature > > > > 5- Wash 3x5 minutes with PBS/tween-20 0.05% > > > > 6- Add 100 µL Envision solution (goat anti-mouse/rabbit) and incubate > for 1hr at RT > > > > 7- Wash 3x 5 minutes with PBS/tween-20 0.05% > > > > 8- Incubate with DAB (1:50) for 10 min (between 5-10 min; check color > development) > > (wear gloves, carcinogenic!) > > > > 9- Rinse thoroughly with miliQ water > > > > 10- Stain with haematoxylin for 1 min > > > > 11- Wash with running tap water for 5 min > > > > 12- Start the alcohol/xylene series (70% EtOH -> 80% EtOH -> 96% EtOH -> > 100% EtOH -> 100% EtOH/xylene -> xylene -> xylene) > > > > 13- Mount slides with entallan > > Kind regards, > > > > > > Esther Kooijman | Research Technician | Department of Radiology and > Nuclear medicine > > The Netherlands > > > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.utsouthwestern.edu_mailman_listinfo_histonet&d=DwIGaQ&c=X2IGR6v8ax_mLhSmU1r3Aw&r=NAc-sZTcJtcdymsLif_fp7ngB_dNBpF-UI64u9fuosc&m=N05bQ_qJ2Li62f83kZl-FK7rp5Ad-spj9JTvwovnnKQ&s=6MYtZ-ICVPPxaohoUjidJjm_mNhwB7nDA1Np5SRxT4w&e= > ___ > 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] Help with frozen spleen and liver tissue!
Hi all, Can someone please provide me with more details regarding cryosectioning of mouse spleen and liver tissue? Currently, I've been fixing my samples in 4% PFA (they are well fixed - I know that will be the first question asked!), and then cryoprotect the tissue in a series of graded sucrose solutions (15% to 30%) until they sink. However, when I go to place the sections on the slide from the cryostat, they look great initially under the microscope, but once they dry they have poor morphology, especially seen in the liver. I've never run into this issue before, with either brain or spinal cord which are significantly more delicate. I've tried cutting the tissue free-floating as well to see if it was how I was placing the sections on the slide, but nothing seems to work. The morphology in the liver is so terribly compromised that I cannot visualize the sinusoids properly. It is baffling that once they go onto the slide they look okay, but 5 minutes later the tissue appears to "separate" from itself. Does anyone work specifically in frozen tissue sections, liver and spleen in particular? If so, would you be able to help me figure out the best way to generate quality specimens? Thank you! Regards, Allyse Mazzarelli ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Help with liver/heart tissues
Good afternoon, I am having a few issues getting nice morphology from both liver and heart tissues. I currently work only with CNS tissues (brain, spinal cord, DRG, etc.) but recently, our research team have become interested in immunostaining some peripheral tissues, including the heart and liver. All tissue [mouse] is perfusion fixed with 4% PFA and then post-fixed in fresh 4% PFA for at least 24 - 48 hours. After fixation, the tissue is cryoprotected in 30% sucrose solution for 48 hours. Once cryoprotected, I then section the tissue at various thicknesses depending on the assay I'm running. Previously I sectioned some heart and liver samples (sectioned both at 20um and at 8um) and ran a few H&Es. Unfortunately, the tissue was so damaged and under-fixed that we scrapped the blocks. This time around, the mouse liver tissue was carefully dissected prior to post-fixation into four quadrants to allow for better PFA permealization. Additionally, we post fixed in 4% PFA for 3 days instead of 2, and the tissue was cryoprotected for 2 days. To my surprise, when I sectioned this tissue on the cryostat, I still noticed severe artifacts. It is very difficult to see nice morphology, and there appears that there was an issue with fixation (in the liver the nucleus is flattened and the sinusoids are not clear, etc. the nuclei in the heart are also more flat and the muscle fibers have separated from one another). The staining was not as bad as the first tissue I had sectioned, but I was still unable to get a nice H&E stain depicting clear nuclear/cytoplasm. These artifacts appeared at both 8um and 20um. Does anyone happen to have nice fixation/H&E staining protocols for both liver and heart? I'd be happy to give an in-depth description of the protocol(s) I'm currently using. Additionally, is there anything that appears I'm doing wrong in terms of perfusion/fixation? Thanks so much! Allyse ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] H&E Protocol for Fresh-Frozen Brain and Spinal Cord
Hi Histonet! I was wondering if anyone could provide me with a reliable H&E protocol for fresh frozen tissue. Currently, we cut our slides on the cryostat and store them at either -20 or -80 (depending on the study director) until they require us to stain for morphology. Typically the tissue isn't always perfectly snap frozen, and often times there are freezing artifacts. However, I would like to preserve as much as the morphology as possible without further damaging the tissue. I have really only had experience staining perfusion-fixed tissue using the following staining procedure: Water rinse Hematoxylin (7211 Richard Allen Scientific) for 30 seconds Water rinse under running tap Clarifier 1 (Richard Allen Scientific) with three quick dips Water rinse under running tap Bluing reagent (Richard Allen Scientfic) for 1 minute Water rinse under running tap 95% EtOH for 30 seconds Eosin-Y with Phyloxine (Richard Allen Scientific) for two quick dips 95% EtOH for 30 seconds 100% EtOH for 1 minute 100% EtOH for 1 minute 3, one-minute exchanges in Clear Rite 3 (a xylene substitute, Richard Allen Scientific) However, today I was asked to run an H&E on a fresh frozen slide. I post fixed in 4% PFA for 10 minutes immediately (not allowing the slide to thaw), and then ran the above procedure as normal. When I examined the slides under the scope, there appeared to be terrible freezing artifacts. The study director happened to think it was both a post-fix and staining issue, so I re-ran the procedure without post-fixing, (per his recommendation) as follows: Immediately into hematoxylin for 30 seconds Water rinse under running tap Eosin for 2 quick dips 95% EtOH for 1 minute 100% EtOH for 1 minute 100% EtOH for 1 minute 3, one-minute exchanges in Clear Rite 3 I saw slightly better morphology, but the director still was not happy with the outcome. I have spent the remainder of my afternoon researching different H&E protocols for fresh frozen tissue, and all seem to be relatively similar to the two mentioned prior. Does anyone out there in Histo-land consistently run H&E on fresh frozen tissue, specifically on slides that have been stored for a while? I work particularly with brain and spinal cord, but we occasionally will cut other tissue and organs. I would like to optimize this procedure as soon as possible. Thank you in advance! Regards, Allyse Mazzarelli ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
