|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.