Re: [Histonet] texture/quality of DAB staining
Your pic isn't on the site yet so I haven't been able to see precisely what you are talking about but I have a few
suggestions to increase staining based on your protocol:
(1) I am assuming from your protocol and desire to counterstain with WG that you are doing cytospins of fresh
(2) Did you try other fixations besides methanol? I find that methanol sometimes can reduce staining for certain
(3) Use 0.3% H202 in H20 for 10 minutes as 3% is harsh for cytospins or fresh tissue.
(4) Add another amplification step such as using a primary, biotinylated secondary and then Strep-HRP. Using a
directly labeled primary will reduce your overall amplification.
(5) You could try Vector's ABC instead of Strep-HRP, your staining will be more intense though be careful for
(6) Are you using DAB+? It is more robust than DAB
(7) Do primary overnight at 4 deg C and potentially increase primary concentration.
I will try to give more comments once I see the picture,
----- Original Message -----
From: Ashley Haines
Date: Friday, May 5, 2006 8:14 am
Subject: [Histonet] texture/quality of DAB staining
> 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
> sensesit'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
> projectionsfrom 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
> protocolfor 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
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