Renal Question

From:"Renault, Peter"

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 where
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 aorta
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 left
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 a
standard peroxidase DAB technique. The sections are dehydrated, cleared and
then mounted in Acrytol

Pierre Renault Tech 3
Foothills Hospital 11th Floor
Calgary Laboratory Services
pager  303-8327  *

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