NBF and renal biopsy immunos

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From:Ian <ib@jeack.com.au>
Date:Sun, 25 Jul 1999 12:42:51 +1000
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The methods I applied to renal biopsies were published in the Journal of
Histotechnology a couple of years ago. The relevent references are :1.
Journal of Histotechnology/ Vol 19, No 2/ June 1996 and 2. Journal of
Histotechnology / Vol 17, No 2 / June 1994.

The only changes to the immuno method is that instead of protease type VIII
I use Dako proteinase K for 2 min prior to the primary antibody.
I also found it advantageous to apply a couple of dilutions of the primary
to sequential sections (on the same slide)ie one dilution at the optimal and
one a bit more dilute in case there was any backgroud staining the results
could be compared. eg for IgA a 1:50 and a 1:100 and for IgG a 1:100 , 1:200
and 1:400. This took care of any fixation and processing variation between
individual specimens. Of course optimal dilutions should be determined in
your own lab as there are many variations in fixation and processing between
institutions. A final point to note is that all my methods were applied to
hand processed tissue! The quality of machine processed tissue was
considered inferior for these small pieces of tissue. Please don't hesitate
to ask if you have any problems and good luck with those stubborn
pathologists who generally resist any sort of change.
Ian Birchall
Melbourne, Australia.

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