Dear histonetters

This is the latest installment in our ongoing heart saga.  I previously
wrote regarding the trashed appearance of paraffin-embedded heart sections I
had prepared, a problem we did not have with commercial sections from
Novagen.  The degree of fixation did not appear to be the problem, as H&Es
on the same sections were fine, I thought it was something in our IHC
protocol after dewaxing.

To solve this, I did an experimet where I dewaxed 8 or 9 slides and put them
through the IHC protocol, removing one slide after each step and staining
with hematoxylin and eosin to check for integrity of the cellular
structures.  Bottom line is, all of the slides looked fine up until the
streptavidin peroxidase step, after which the slides again looked chewed.
We use biotinylated secondaries and ready-to-go streptavidin peroxidase from
Zymed.  We incubate with streptavidin peroxidase for 10 mins at room
temperature.

Can anyone suggest what I might be doing wrong and why we dont see this with
commercially prepared slides?  Same results with new and old streptavidin
peroxidase so it has not "gone off".

Many thanks
Gillian





 
Gillian Barlow, PhD
Postdoctoral Fellow
Laboratory of Julie Korenberg, PhD, MD
Cedars-Sinai Medical Center
Davis Bldg, Lab 2007
110 George Burns Rd
Los Angeles, CA 90048

Phone: (310) 423 7650
Fax: (310) 423 0302