I'm still stickin' with the endogenous biotin.
Amos

----- Original Message -----
From: "Renault, Peter" 
To: "'Amos Brooks'" ; "Renault, Peter"
; 
Sent: Monday, August 27, 2001 9:48 AM
Subject: RE: Renal Question


> I apologize for not  stating what the artifact looks like. The artifact
> appears to resemble a dark brown precipitate, every similar to formalin
> pigment, in the brush border area. Your responses have come so quickly,
that
> I truly appreciate the help and the many ideas to pass on to the renal
> department. Thank you so much for your timely assistance here.
>
> Pierre Renault Tech 3
> Foothills Hospital 11th Floor
> Calgary Laboratory Services
> *403-670-4855
> pager  303-8327  *peter.renault@cls.ab.ca
>
>
> -----Original Message-----
> From: Amos Brooks [mailto:amosbrooks@home.com]
> Sent: Friday, August 24, 2001 7:35 PM
> To: Renault, Peter; HistoNet@pathology.swmed.edu
> Subject: Re: Renal Question
>
>
> Hi,
>     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
> blank).
> Amos Brooks
>
> ----- Original Message -----
> From: "Renault, Peter" 
> To: 
> 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
> 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
> > *403-670-4855
> > pager  303-8327  *peter.renault@cls.ab.ca
> >
> >
>