[Histonet] Help with muscle
Hello All, I hope I am not asking a dumb question. As I know that everyone on the histonet is very knowledgeable I was hoping to get some suggestions regarding processing tissue for neuromuscular junction staining. We are a neuro research lab and want to quantify neuromuscular junction on treated and non-treated rat gastrocnemius. We need the whole muscle for quantifications. The first problem we encountered when attempting to do this on PFA perfused and post fixed whole muscle was negative staining with SV2 antibody (presynaptic) but nicely stained alpha bungarotoxin (post synaptic) end plates. After a few attempts with no success we decided to freeze the whole muscle in dry ice and isopentane. First off we are having issues with the middle of the muscle freezing completely even after leaving it in solution for more than 10 min. Secondly while we do get nice staining with SV2 and bungarotoxin in some of the endplates we don’t see colocalization in most of the NMJ’s. One possibility that I was thinking could be causing this is that when the muscle is being picked up on the slide (cryosections) it is not laying completely flat on the slide (we tend to have to focus back and forth quite a bit) so we see the SV2/bungarotoxin near each other but not overlapping. One other thing I thought might be occurring is that when the muscle is being frozen it is retracting causing a shift in the presynaptic/postsynaptic NMJ. What is the best way to process the tissue, fixation or freezing? Any suggestions are greatly appreciated. Thanks, Leslie Leslie Garcia Senior Histologist Clive Svendsen Lab Board of Governors Regenerative Medicine Institute Cedars-Sinai Medical Center 8700 Beverly Blvd. AHSP 8405 Los Angeles, CA 90048 Phone - 310-248-8571 Web - http://www.cedars-sinai.edu/RMI ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
FW: [Histonet] help with muscle stains
Angela, Martin, T.P. et al 1988, Am. J. Physiol. 255 C43-50 I use this published technique for SD and GPD, works a treat. If you cannot get the journal let me know and I'll forward the protocol. Ian. Dr. Ian Montgomery, Histotechnology, I.B.L.S. Support Unit, Thomson Building, University of Glasgow, Glasgow, G12 8QQ. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Angela Bitting Sent: 16 March 2009 04:10 To: histonet Subject: [Histonet] help with muscle stains Would anyone like to share their procedures for Cytochrome oxidase(COX), Succinic dehydrogenase (SDH) and NADH-TR with me? I have old, old ones but they are sketchy. Thanks. Angela Bitting, HT(ASCP) Technical Specialist, Histology Geisinger Medical Center 100 N Academy Ave. MC 23-00 Danville, PA 17822 phone 570-214-9634 fax 570-271-5916 No trees were hurt in the sending of this email However many electrons were severly inconvienienced! IMPORTANT WARNING: The information in this message (and the documents attached to it, if any) is confidential and may be legally privileged. It is intended solely for the addressee. Access to this message by anyone else is unauthorized. If you are not the intended recipient, any disclosure, copying, distribution or any action taken, or omitted to be taken, in reliance on it is prohibited and may be unlawful. If you have received this message in error, please delete all electronic copies of this message (and the documents attached to it, if any), destroy any hard copies you may have created and notify me immediately by replying to this email. Thank you. BEGIN:VCARD VERSION:2.1 X-GWTYPE:USER FN:Bitting, Angela TEL;WORK:570-271-6844 ORG:;Histology EMAIL;WORK;PREF;NGW:akbitt...@geisinger.edu N:Bitting;Angela END:VCARD ___ 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 muscle stains
Would anyone like to share their procedures for Cytochrome oxidase(COX), Succinic dehydrogenase (SDH) and NADH-TR with me? I have old, old ones but they are sketchy. Thanks. Angela Bitting, HT(ASCP) Technical Specialist, Histology Geisinger Medical Center 100 N Academy Ave. MC 23-00 Danville, PA 17822 phone 570-214-9634 fax 570-271-5916 No trees were hurt in the sending of this email However many electrons were severly inconvienienced! IMPORTANT WARNING: The information in this message (and the documents attached to it, if any) is confidential and may be legally privileged. It is intended solely for the addressee. Access to this message by anyone else is unauthorized. If you are not the intended recipient, any disclosure, copying, distribution or any action taken, or omitted to be taken, in reliance on it is prohibited and may be unlawful. If you have received this message in error, please delete all electronic copies of this message (and the documents attached to it, if any), destroy any hard copies you may have created and notify me immediately by replying to this email. Thank you.BEGIN:VCARD VERSION:2.1 X-GWTYPE:USER FN:Bitting, Angela TEL;WORK:570-271-6844 ORG:;Histology EMAIL;WORK;PREF;NGW:akbitt...@geisinger.edu N:Bitting;Angela END:VCARD ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] help with muscle histology
Fellow Histonetters...any help would be appreciated. I have a couple questions about getting good muscle samples. I have a project in the lab right now that is working with laser. The student is taking rat legs for this. What he does is cut a laser line into the large muscle of the rat lag at different settings. Then he sacrifices the rat and harvests the entire leg. He fixes this (this time fro 3 weeks) so I know it is fixed and then cuts the sample just before and after the laser cut. Here are the tweo problems I need help with: 1. When he does this he leaves the rat femur in the sample. So, I have been decaling this with an EDTA solution. Should I use something else? 2. When I cut these after they have decaled and been processed etc., the muscle seems to separate instead of staying closely bundled. This is a problem as we want to measure the zone of tissues damage from the laser mark. 3. Any help would be appreciated..I thought maybe you could give me would be greatly appreciated!! Thanks in advance. Colleen Forster U of MN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
