RE: help required

From:Young Kwun

I have some experience in using Zamboni's fixative a long time ago. I agree with Ms Bonnie Whitaker saying that serial sections might be better or you might try frozen sections (after fixation) just to make sure your method is working. I only used ovine oesophagus tissue, so I had to use thick frozen sections instead of whole mount preparation in studying myenteric plexuses.
The methodology of the whole mount preparation and immunostaining has been well established during the 1980's. It would be very helpful if you look up some papers by Drs Furness JB and Costa M form the Flinder's University in South Australia during that period. They mainly used guinea-pig small intestines which is much thicker than your tissue I guess. After using Zambonie's fixative, I had no problem with immunostaining with the antibodies you mentioned including CGRP, VIP, Substance P and Neuropeptide Y etc.
Best wishes

Young Kwun
Senior Hospital Scientist
Dept. of Anatomical Pathology
Concord Hospital
Concord NSW 2139 Australia

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-----Original Message-----
From: Kid'surgeon * []
Sent: Tuesday, 17 December 2002 16:57
Subject: 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 u suggest something better?


Marry Christmas

Parkash Mandhan
*chch, nz.*
>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.
>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 * []
> 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.
> 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
> Parkash
> *chch, nz.*
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