[Histonet] Help with muscle

[Jennifer MacDonald]

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