Re: Renal Question

From:Amos Brooks

    Not seeing the artifact myself I am inclined to think that it might be
Endogenous Biotin. It is notorious in kidney and particularly in the brush
boarder of the tubules. Try a labeled polymer like Envision from DAKO or
Mach2 from Biocare Medical (I am sure there are others but I am drawing a
Amos Brooks

----- Original Message -----
From: "Renault, Peter" 
Sent: Friday, August 24, 2001 11:05 AM
Subject: Renal Question

> Good Day fellow Histonetters, I was asked a question in regards to a
> precipitate which is forming on the brush boarder in the rat kidney tissue
> being processed here at out institution. I am stumped as to the cause so I
> have copied and pasted the information in hopes someone else may have come
> across this before. I appreciate any input you may have to alleviate this
> problem. Please read the information below to see if you can determine
> the problem lies and how to best remove the precipitate from the brush
> boarder which is interfering in the final interpretation. Thanks again in
> advance for your help.
> The procedure calls for normal rat kidney's to be perfused through the
> with methyl Carnoy's or 4% buffered paraformaldehyde. Transverse kidney
> slices are dissected and immersed for 60 minutes in the same fixative on
> ice, and placed at 4C in ethanol overnight. The tissue is then processed
> without formaldehyde and embedded in paraffin. 4um sections are cut and
> to dry overnight. These are then dparaffinized, hydrated and subjected to
> the following treatments with 2 x 3 minute washes of PBS ( ph.7.4) between
> each step.
> 1. 4.5% hydrogen peroxide in methanol for 10 minutes
> 2. Avidin solution for 15 minutes
> 3. Biotin solution for 15 minutes
> 4. 10% normal ( non-immune) goat serum for 10 minutes
> The goat serum is not washed off. The primary antibody is added to it and
> the specimen is incubated overnight at 4C. The antigen is visualized with
> standard peroxidase DAB technique. The sections are dehydrated, cleared
> then mounted in Acrytol
> Pierre Renault Tech 3
> Foothills Hospital 11th Floor
> Calgary Laboratory Services
> *403-670-4855
> pager  303-8327  *

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