Hi, Judy

I am not sure if I can help since I have never worked with bone nor methyl
methacrylate.  But, I have done BrDU immuno (on NBF fixed, paraffin embedded
coronary arteries) and can tell you that it required a lot "destructive"
pre-immuno steps including 15 to 20 minutes in 1.5N HCl, 5 minutes in 0.1M
Borax, and 7.5 minutes in prewarmed 0.1% trypsin at 37 degrees C, with
appropriate washes in between.  Also, the primary abody incubation was 2
hours at RT.  I did not set up this protocol, but I was told that it took a
lot of tweaking to get it to work.  The timings need to be pretty much
exact.   

Don't know if this helps.  Good luck!

C
 

-----Original Message-----
From: Judy Strauss [mailto:straussj@upstate.edu]
Sent: October 4, 2000 11:13 AM
To: histonet@pathology.swmed.edu
Subject: Miracles Needed


Dear Histonetters,

I have inherited a project of 2% paraformaldehyde/2%
glutaraldehyde/ruthenium red fixed rat tibia embedded at low temperature in
MMA.  I hope someone out there is immunostaining bone and can give me
advice.

Basically, I need two miracles:  
1)  BrDU   Can anyone tell me why I can not retrieve this antigen in MMA? 
(I have tried steam, boiling citrate
buffer, autoclave, pepsin digestion)   A couple specimens were embedded in
epon/araldite and can be successfully stained with a Becton-Dickinson
primary antibody.   
2)  TGF-beta1   Can anyone suggest a primary antibody for TGF-beta1 to be
used in this same rat tibia tissue?

As you can see I really need your collective experience.  Thanks in
advance.

Judy Strauss
Institute for Human Performance
SUNY Upstate Medical University
Department of Orthopedic Surgery
Syracuse, New York