[Histonet] p16 Nordi QC
There is a fantastic resource for antibody assessment and optimization and they have done p16 as well as many many other antibodies. Their results of testing is free on line. p16 is http://www.nordiqc.org/Epitopes/p16/p16.htm from 2009 with many different companies antibodies. Here is the list of their epitopes http://www.nordiqc.org/epitopes.htm Donna Harclerode, HT, ASCP, HTL, QIHC Lead Histotechnologist VA San Diego, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Anti static gun
Available through VWR, but may be cheaper elsewhere. They work much better than I expected. ZEROSTAT ANTISTATIC GUN VWR cat #100496-120 Vendor cat #60610 Each$122.64 list Donna Harclerode, HTL, QIHC, SLS (ASCP) Associate Scientist Celgene 9393 Towne Center Road San Diego, CA 92121 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Trichrome stain on frozens
I had major problems with frozen trichrome years ago and Jamie Watson from GNF, La Jolla said to fix in the Bouins and not fix in formalin and then mordant in Bouins. (duh) I am not sure why I never tried that, but when I fixed in Bouins for 10 minutes I got consistent trichrome on frozen skin sections. Donna Harclerode, HT,HTL (ASCP) QIHC, SLS Arista Molecular Immunohistochemist 10455 Pacific Center Court San Diego, CA 92121 858-866-5421 donna.harcler...@aristamolecular.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Folding sections
Hi Alexia I am not sure exactly what you are describing but my first question would be what is your nuclear stain in? If it is an alcoholic solution the sections may get shocked and stick together. I normally used DAPI in the secondary to counter-stain nuclei which is aqueous so that was less stress on the slices. I put 1-3ml of the 0.3% Triton in the mounting dish- it helped flatten and drag the slides on to the slides. For incubations I used micro centrifuge tubes with VERY slow rotation. Primary was ON at 4o. Rinses were in dishes from Brain Research. We changed the nets to panty hose and tightened them and glued them down with nail polish. I usually was staining 30u sliding microtome sections not vibrotome so may not cross over. I tried not to use paint brushes- they tended to damage the sections due to sticking to my sections. I made bent glass rods out of pasture pipettes with a bunsen burner. I once had the knife angle too steep on the sliding microtome, cut too fast and then had curled sections that never did flatten right. I do not know if that is is a possibility on the vibrotome. Good luck Donna Harclerode HT,HTL,QIHC (ASCP),SLS Histology Core Manager UCSD, Dept of Pathology 9500 Gillman Drive BSB 2009 San Diego, CA 92093 858 534 7438 Date: Wed, 29 Sep 2010 22:14:31 + From: Alexia Francisca N??ez Parra alexia_f...@hotmail.com Subject: [Histonet] Free floating sections To: lita de foro histonet@lists.utsouthwestern.edu Message-ID: snt129-w2203a4dc1629c55f3b778e93...@phx.gbl Content-Type: text/plain; charset=iso-8859-1 Hello Histonetters! I am currently running a immunofluorescence reaction using free floating brain sections and am having problems with the sections folding as I lift them to mount at the end of the reaction. To obtain the sections, I first perfused the mouse with 4%PFA, dissected the brain and postfixed it in 4%PFA for three hours, washed it twice in PBS solution, and stored the brain in PBS at 4 degrees celcius. Then, I used a vibratome to slice the brain in 100um sections (I have also tried using a cryostat - and tissue tek- but this immuno runs better on sections from the vibratome). After being sliced, the sections were stored at 4 degrees in PBS. I am running the immuno using a culture plate with incubation with a blocker (10%DS in PBS-T), primary antibody incubation overnight at RT, and incubation with secondary antibody at RT for two hours. All these incubations are made under agitation. Also, I am co-staining the cells with the nuclear stain TOPRO. To mount the sections, I am lifting them using a thin paintbrush and placing them into a petri dish filled with PBS. Then I drag the sections on to a slide, apply Vectashield and the coverslip. Normally the sections should unfold as soon as they are placed in PBS solution, but many of them are staying folded on the brush. I have already tried incubating the primary antibody at 4C and I observed even more folding. Can you suggest anything to prevent this? Thank you very much Alexia Nunez-Parra Graduate Student University of Maryland ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Flat mount eye sections
I have done flat retina sections- which may be what you are being asked to do which is a very specialized procedure even for most research labs. Do you have access to a sliding microtome with a freezing stage? Otherwise I do not know any way to do this. If you are doing flat retina (I have done birds and rats- the rats are very difficult and require a dissecting scope to even prepare them). I did this in a lab 15 years ago but now that I am older and do not see so well, I would use a scope for any species. For birds or rodents, perfuse the animal usually 4%PFA, remove the eye, excise the cornea, iris and lens and being carefully not disturbing the retina remove the vitreous humor and post fixed in 4% at 4oC overnight Transfer to 30% sucrose in PBS at least another 24 hours Dissect the retina from the eyecup and trimmed it with a fresh scalpel blade to flatten it out. I cannot remember the correct term, but pigeon and chicken eyes have a comb structure inside their eyes that must be removed before flattening. It usually took about 4 cuts of varying sizes to get a flat sample. Looks kind of like an uneven flower with cuts from the center out. On a few eyes they had to be cut into 2 pieces to get them flat. On a sliding microtome with freezing stage (or with dry ice on either end of the platform, build a 30% sucrose platform bigger than the eye to be sectioned Then with the blade in place, shave down the platform to make a flat place big enough to mount the retina. Remove the blade for safety reasons for mounting of the retina. Use a wooden block that is super flat covered with saran wrap. Very carefully on one side so there were no wrinkles in the wrap, place the inside of the eye against the saran wrap (with no liquid or it will slide off) and very carefully but quickly wipe the retina onto the prepared platform and let it freeze. Replace the blade from and drop the stage or raise the blade (depending on the microtome ) and carefully section the retina. On a pigeon we usually got at best 3 30u sections but usually did not get the retina quite flat and got 4 sections. The sections are collected in PBS and stained in micro centrifuge tubes for various antibodies then mounted on slides after staining.. Good luck Donna Harclerode HT,HTL,QIHC (ASCP),SLS Histology Core Manager UCSD, Dept of Pathology 9500 Gillman Drive BSB 2009 San Diego, CA 92093 858 534 7438 Date: Wed, 04 Aug 2010 10:57:02 -0700 From: sgoe...@xbiotech.com Subject: [Histonet] Flat Mount To: histonet@lists.utsouthwestern.edu Hello all, I am being asked to do immunostaining on fla eyes. First, what is a flat mount and does an on how to make one? Second, what is the methodol stains on these? Do you have to fix them, cut them,done this and need some help. Thanks guys!! Sarah Goebel, B.A., HT (ASCP) Histotechnician [DEL: XBiot 8201 East Riverside Dr. Austin, Texas ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Tungesten Carbide Knives and araldite
I have a tiny project using araldite on mouse nerves and wondered if anyone could recommend the tungsten carbide knife that Ted Pella cat #121-50 or any other company sells. I cut methacrylate ages ago with glass knives but wonder if the tungesten carbide blade might be a easier and way less expensive option than a $9k Leica glass knife breaker. I used the Sorvall breaker but it looks to be discontinued. If anyone has an opinion on araldite, I would appreciate that too. Thanks Donna Donna Harclerode HT,HTL,QIHC (ASCP),SLS Histology Core Manager UCSD, Dept of Pathology 9500 Gillman Drive BSB 2010 San Diego, CA 92093 858 534 7438 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] San Diego Chapter meeting
The San Diego chapter will be having our next meeting on Sept 10 from 4:30 to 7 at Pfizer. The topic will be on laboratory safety and hopefull we will come up with more topics for future meetings. There will be a light dinner and everyone is welcome but please let us know you are planning to attend. Please contact me for further information or to RSVP Thanks Donna Harclerode erid...@cox.net ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Cryostat thick and thin sections
One more possible issue along with the others. Over tightening of some disposable blades can cause thick and thin sections. Leica 1800 and 3050 only should have the knife tightened snug, not have the handle (tightening bar or whatever you call it) pushed as far as it goes. My rule of thumb is to go just beyond the angle of the knife and quit there. I love the 3050- it is my favorite for any cryo work. Donna Harclerode HT, HTL, (ASCP),QIHC, SLS ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
