[Histonet] FW: BrdU

[Bruijntjes, J.P. (Joost)]

Thanks to those with an answer, problem is solved. That means that one of the pretreatment steps (pronase) stopped working. Changing to another batch pronase was enough to see BrdU staining in both rats and mice

 

Thanks agian

 

Joost 

 

________________________________

From: Bruijntjes, J.P. (Joost) 
Sent: donderdag 11 september 2008 12:04
To: histonet <@t> lists.utsouthwestern.edu
Subject: BrdU

 

Hi Histonetters

 

I do have a problem with BrdU, and I hope someone of you can give a kind of solution/explanation.

 

I work with NALT (Nose Associated Lymphoid Tissue) of both rats and mice, which were fixed in a fixative composed of acetic acid, formaldehyde, ethanol and aqua dest. After 48 hours this fixative was replaced by ethanol. Later on the tissues were dehydrated and embedded in paraffin. Just because the lymphoid tissues material is so small, about 10 paraffin slides were collected.

I started with the rat NALT's. One paraffin slide was stained with HE, and a few other slides were stained with a monoclonal antibody directed against BrdU. I was satisfied with the staining, so far no problem.

 

After the rats, the same story with the mice NALT's. But I did not get any positive staining. Some days later I repeated the first try-out included with some positive controls from the rat-study with the primary antibody I used in the first part, and the biotin conjugated primary antibody as well, but again no positive staining. The pre-treatment of the slides for rat and mice is the same. The only difference lies in the primary and secondary antibody. 

 

For rat tissue I use a non-conjugated monoclonal antibody, followed by HRP-conjugated powervision.

For mice tissue I use a biotin conjugated monoclonal antibody, followed by a HRP-conjugated streptavidin.

 

Can anyone give me an explanation? The storage of the slides was in a room with an equal temperature (about 20-21ºC) and humidity. 

Is it possible that the epitopes in the single slides are destroyed so that they are not recognizable anymore by the antibody? The time between preparing the slides and the BrdU staining of the rats NALT slides was at the most 4 weeks, while the first negative staining on the mice NALT's appeared after about 8 weeks. 

 

Thanks in advance

Joost Bruijntjes

TNO Quality of Life

Zeist

The Netherlands

    

 

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Joost Bruijntjes

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