[Histonet] texture/quality of DAB staining

From:"Ashley Haines"

Hi folks,

 

I am posting a picture called "fine DAB staining"
(http://www.histonet.org/site_images_frame.asp).  I am hoping for
suggestions on how to improve the quality of the staining.  In some senses
it's a great result.  You can see, at least under the scope if not in the
posted micrograph, lots of fine detail.  There are many fine projections
from the cell surface which stain positive.  Unfortunately, my goal is to
counterstain with a Wright Geimsa-like or Hema3 stain.  The signal I am
getting will not be visible with that counterstain.  Can you suggest any
ways to make my staining coarser and/or darker.  Here's my basic protocol
for the posted micrograph:

 

MeOH fix cytospin, block with 3% H202 in MeOH 1hr at RT, wash, block with
10% goat serum 1 hr, wash, incubate with biotinylated primary 1hr at 37,
wash, incubate with SA-HRP (1:1000) for 1 hr at RT, wash, detect with
Vector's DAB substrate kit (with nickel enhancement) 20 min at RT, wash in
DI.

 

Thanks!

Ashley

 

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