Re: help required

From:Kid'surgeon *

thanks for reply.

yes i do my washes on shaker for 10-15 min in 15-20 ml of BPS (min 3)

u mean i wash tissues after primary and sec with 1% triton in PBS?


Parkash
cirgien4babys
*chch, nz.*

 

>From: Kathleen Spencer

>To: Kid'surgeon *
>Subject: Re: help required
>Date: Mon, 16 Dec 2002 15:18:52 -0600
>
>I would try adding triton to your PBS washes after antibody. I use a
>.1% , but I have 20 micron sections. 1 mm is really thick, but it
>might work. Are you shaking the tissue on a shaker?
>
>Kathleen Spencer
>On Monday, December 16, 2002, at 02:42 PM, Kid'surgeon * wrote:
>
>>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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