help required

From:Kid'surgeon *

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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