RE: help required

thanks for reply.

i am staining one antibody at one time.

DMSO is to wash out the zamboni and enhance penetration.

i use 1% triton-X, what lower percentage do u suggest me to use?

I am using whole mount tech as i am looking for the enteric nervous sytem. It gives a paramount view of the gur musculature and one can nicely c the whole nerve plexues beautifully, which i guess is very much impossible to get in sections.....my view

i have not tried other fixatives....may be need to switch from zamboni to these at some stage...

have someone in the group used counterstains for this this problem of autofluorescence? if yes, what and can he/she send me the ref for methods etc

cheers

Parkash Mandhan

cirgien4babys

*chch, nz.*

>From: "PMarcum"

>To: "Kid'surgeon *"

>Subject: RE: help required

>Date: Mon, 16 Dec 2002 16:14:23 -0500

>I am sorry but this really needs clarification. Are you attempting to stain

>the entire plexus at once with all of the antibodies at once? They are

>generally used one at a time on different pieces of tissue unless you are

>double labeling with a second chromogen/2nd antibody system. Why are using

>DMSO and at what percentage? (I understand the penetration issues, this just

>sounds like overkill when you include the penetration rate of Zamboni's.)

>The background could come from using everything at once. The Triton X-100

>percentage is very high and should be lowered as it can even penetrate and

>explode or implode fixed cells at that concentration. Sections for IHC are

>generally sectioned at 4 to 6 microns not one millimeter. The depth of the

>tissue would make viewing almost impossible. Picric acid may be part of

>your autofluorescence problem also. Have you considered a more traditional

>10% NBF or 4% paraformaldehyde? Overnight in Zamboni's may be too much.

>This is a rapid fixative and could be rough on fetal tissues. Pam Marcum

> -----Original Message-----

> From: Kid'surgeon * [mailto:childoc@msn.com]

> Sent: Monday, December 16, 2002 3:43 PM

> To: histonet@pathology.swmed.edu

> 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.

> The summary of tech is:

> Fetal rats tissues are fixed in Zamboni (overnight at 4C)

> Put in to DMSO ( 3-changes)

> Washed in PBS ( 3-changes)

> The gut layers rseparetd and muscular layer is preserved for looking of

>myenteric plexus. The thichness of tissue will be 1 mm (app).

> Put into 10% Goat Serum and 1% triton X-100 in PBS for ONE hour in Humid

>chamber

> Washed with PBS ( 3-changes)

> Primary antibodies r added (NSE, VIP, SP100 and CGRP from rabbit) and

>incubated for 16-18 hrs

> Washed in PBS ( 3-changes) on shaker

> Sec antibody (anti-rabbit conjugated with FITC) from Goat is added and

>speciemen incubated for 120 min in humid chamber

> washed with PBS ( 3-changes) shaker

> Mounted and slides r views under fluorescent microscope.

> With this techniques, i am having following problems and need some

>technical help from u.

> 1. High background (tissue is not autofluorescent)

> 2. really cann't see and appreciate the nerve plexues.

> I will appreciate the technical guidance.

> cheers and HAPPY CHRISTMAS to u all

> Parkash

> *chch, nz.*

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