[Histonet] texture/quality of DAB staining

[Andrea Hooper]

Hi Ashley,

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 
cells????
(2) Did you try other fixations besides methanol? I find that methanol sometimes can reduce staining for certain 
antigens?
(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 
background
(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,
Andrea

----- Original Message -----
From: Ashley Haines <ahaines <@t> vims.edu>
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
> DI.
> 
>