RE: help required (IHC)

From:Andrew Gray

Hi Parkash,

I'm doing very similar immuno work to you in my PhD at the moment
(mature rats though).   (Perhaps one of the pessimists out there could 
mark my thesis?)

I would suggest getting a hold of a primary Ab that is known to 
reliably and intensely label the nerves.   I had a similar problem 
until I obtained some anti-calretinin Ab from a nearby lab.   This 
validated my technique and method and showed very dramatically the 
layout of the plexus.   Another nerve stain is anti-PGP 9.5, and I find 
the nerve nucleus stain anti-hu to work quite well.    

Your original procedure seems fine, I use 3 x 10 min washes in PBS, no 
detergent or retrieval required.   My myenteric plexus whole-mounts are 
closer to 0.1mm in thickness (tissue originally stretched out with pins 
on balsa wood).   You could always incubate with primary Ab for a 
longer period of time.   What mounting medium are you using?   Have you 
removed the adherent circular muscle (requires beta blockers to 
stop 'the shakes')?   Once you have done that the myenteric neurones 
should be at the surface.   I would encourage you to continue with 
whole-mounts as you get the whole plexus whereas with sections it's 
touch-and-go what nerves you hit, if you hit them at all.

Best of luck

Andrew Gray
Victorian College of Pharmacy
Monash University

> -----Original Message----- 
> From: Kid'surgeon * [] 
> Sent: Monday, December 16, 2002 2:43 PM 
> To: 
> Subject: help required 
> I am a clinician and doing a year of research. I am doing 
>imuunhistochemical study in the enteric nervous system of fetal rats. 
I am 
>using whole mounts for this study. 

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