RE: help required
Hi,
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
Young Kwun
Senior Hospital Scientist
Dept. of Anatomical
Pathology
Concord Hospital
Concord NSW 2139
Australia
Tel)61-2-9767-6075
Fax)61-2-9767-8427
kwuny@email.cs.nsw.gov.au
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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.*
>
>
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