[Histonet] sections lifting along the edges
Hello, I have a problem with sections which lift off the slides. The slides are APTES coated and it isn't the entire section lifting off the slide, just the peripheral bits. I did not prepare the tissue nor section it. I am told the rat was killed and perfused with PFA for 10 minutes and then the brain removed and dehydrated in sucrose. It was NOT postfixed overnight in PFA. The dehydrated brain was then set in tissue tech and sectioned on a cryostat. 40 micron sections were made. These sections are lifting along the periphery. What is more is that antibody staining is not consistent through the sections and antibody penetration is only about 4 microns. Various conflicting reasons are given for this lifting and antibody inconsistency problem. The histologist suspects that the tissue was either not fixed enough or the the sections were not left to adhere to the APTES slide for long enough before being frozen at -20 oC. The one prof things the tissue was over-fixed. One of the antibodies we are using is the cannabinoid receptor CB1 antibody. Our signal is really poor and bleach very quickly. We found an article where the tissue was postfixed (presumably in PFA) over night and the staining is excellent... The person who did the section reported that she saw what looked like ice-crystals in the tissue. I wonder if this could be a sign that the fixation was not homogenous, i.e. the tissue now had a different texture which made it look like ice crystals. I also wonder about the -80 oC freezer which was used. It is very full and so probably serving more as an insulator than a rapid freezer. As such freezing may have been slow and ice-crystals could have formed but I don't really see tissue damage typical of freezing and the sections life along the periphery not the center... Any ideas on what is happening would be much appreciated. The person I am working with still has rats to kill and we would like to be sure that their brains will be fixed properly and not go to waste. Thanks -- Tyrone Genade http://tgenade.freeshell.org email: tgen...@gmail.com tel: +27-84-632-1925 (c) Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord. To find out how to receive this FREE gift visit http://www.alpha.org. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] AUTO: Laura Miller is Out of the Office. (returning 11/08/2011)
I am out of the office until 11/08/2011. I am on vacation until Tuesday. I will respond to your message when I return. Note: This is an automated response to your message Histonet Digest, Vol 96, Issue 9 sent on 11/5/2011 9:41:14 AM. You will receive a notification for each message you send to this person while the person is away. __ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email __ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Help
I am currently a histology student at Keiser University. I am doing a project for my routine staining class about Celestine Blue. I've been able to find information on why it was created, the chemical make up, and some of it's uses including the trichrome stain. I am having trouble finding images of slides stained with Celestine Blue. Any additional information about the uses would be helpful as well! Thank you, Corrinne VernickKeiser UniversityFL U.S.A ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] sections lifting along the edges
I would fix the profused fixed tissue in formalin for 12-24 hours before infiltrating it in 30% sucrose then snap freeze it, the researcher who thinks it is over fixed is wrong and that is very common, researchers think that aldehyde fixations inhibitis IHc staining but things have changed and especially with antigen retrieval techniques it is better to fix and then infiltrate in sucrose and snap freeze to get the best results, profuse fixation for 10 min is not enough. Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tyrone Genade Sent: Saturday, November 05, 2011 3:03 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] sections lifting along the edges Hello, I have a problem with sections which lift off the slides. The slides are APTES coated and it isn't the entire section lifting off the slide, just the peripheral bits. I did not prepare the tissue nor section it. I am told the rat was killed and perfused with PFA for 10 minutes and then the brain removed and dehydrated in sucrose. It was NOT postfixed overnight in PFA. The dehydrated brain was then set in tissue tech and sectioned on a cryostat. 40 micron sections were made. These sections are lifting along the periphery. What is more is that antibody staining is not consistent through the sections and antibody penetration is only about 4 microns. Various conflicting reasons are given for this lifting and antibody inconsistency problem. The histologist suspects that the tissue was either not fixed enough or the the sections were not left to adhere to the APTES slide for long enough before being frozen at -20 oC. The one prof things the tissue was over-fixed. One of the antibodies we are using is the cannabinoid receptor CB1 antibody. Our signal is really poor and bleach very quickly. We found an article where the tissue was postfixed (presumably in PFA) over night and the staining is excellent... The person who did the section reported that she saw what looked like ice-crystals in the tissue. I wonder if this could be a sign that the fixation was not homogenous, i.e. the tissue now had a different texture which made it look like ice crystals. I also wonder about the -80 oC freezer which was used. It is very full and so probably serving more as an insulator than a rapid freezer. As such freezing may have been slow and ice-crystals could have formed but I don't really see tissue damage typical of freezing and the sections life along the periphery not the center... Any ideas on what is happening would be much appreciated. The person I am working with still has rats to kill and we would like to be sure that their brains will be fixed properly and not go to waste. Thanks -- Tyrone Genade http://tgenade.freeshell.org email: tgen...@gmail.com tel: +27-84-632-1925 (c) Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord. To find out how to receive this FREE gift visit http://www.alpha.org. ___ 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
