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?
>From: "Bonnie P Whitaker"
>To: "Kid'surgeon *"
>Subject: RE: help required
>Date: Mon, 16 Dec 2002 18:09:05 -0600
>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
>Sorry to be such a pessimist, but good luck anyway.
>University of Texas Medical School at Houston
>6431 Fannin Street
>Houston, Texas 77030
> -----Original Message-----
> From: Kid'surgeon * [mailto:email@example.com]
> Sent: Monday, December 16, 2002 2:43 PM
> To: firstname.lastname@example.org
> 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
> 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
> *chch, nz.*
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