RE: help required

thanks for ur reply. Since i am using the whole mount technique, the thickness will be variably more or less same. I can't do anything for that and this study will have only "results" with WMP than with sections. I use washout for 10-15 min in 15-20 mls of PBS on shaker. Would think to add detergents in washout....do u suggest something better?

cheers

Marry Christmas

Parkash Mandhan

cirgien4babys

*chch, nz.*

>From: "Bonnie P Whitaker"

>Reply-To:

>To: "Kid'surgeon *"

>Subject: RE: help required

>Date: Mon, 16 Dec 2002 18:09:05 -0600

>Hi!

>You are very brave!! I don't have any experience with this type of thing,

>but I can't imagine that you would get good staining at 1mm thick. Even if

>stuff gets in, you're going to have a heck of a time washing in-between

>steps. Is there any way that you can do this on serial sections that are

>thicker than normal, perhaps? My only other suggestion is to use lots of

>detergent in the washes. You didn't state how long the washes were, but in

>this case, I suggest the longer the better. Formaldehyde pentrates tissue

>at the rate of ~1mm hour.... these molecules are MUCH larger, so it will

>take longer going in, and longer coming out.

>One other thing, Zamboni's hasn't been used that much for IHC.... I would

>question whether or not to use it as my fixative for an immunostaining

>project.

>Sorry to be such a pessimist, but good luck anyway.

>Bonnie Whitaker

>University of Texas Medical School at Houston

>6431 Fannin Street

>MSB 2.234

>Houston, Texas 77030

>Phone 713.500.5363

>Fax 713.500.0733

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

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

> Sent: Monday, December 16, 2002 2:43 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.*

>----------------------------------------------------------------------------

>--

> The new MSN 8: smart spam protection and 2 months FREE*