Re: [Histonet] Background with DAB staining

From:Gayle Callis

We do CD11c antibody all the time on frozen sections, and do not get 

First, cut your sections at 5 um instead of 10 um, 6 um is ok too. Pick up 
your sections on Silane coated or plus charged slides or poly L lysine 
coated but NOT gelatin coated.  You can prepare these slides yourself, 
HISTONET has methods to use, do a search in Histonet Archives

Air dry sections overnight, then fix in an acetone alcohol mixture at RT 
for 5 min then go directly to 3 changes buffer.  Do NOT air dry 
again.  Proceed with staining.   Our staining buffers whether we use either 
TRIS buffered saline OR Dulbeccos PBS (Sigma) contain 0.2% goat serum and 
0.05% Tween 20.  All diluents, normal serum blocks are made up in this buffer.

We prefer the Glucose oxidase peroxidase block for cleaning up endogenous 
peroxidase particularly for dendritic cell staining, if you want - I will 
attach to you privately.  Most of the time we have success with DAKO 
peroxidase blocker, S2001 for 10 - 15 minutes, we do not follow their 
instructions for 5 minutes, and it has NOT hurt our staining but does a 
more efficienct job of quenching endog peroxidase.

On thing to remember, CD11c is a clone from Armenian hamster and you must 
use a secondary that detects the Armenian hamster, and the secondary MUST 
be adsorbed to the mouse.  There are two sources of secondaries we use, our 
favorite is Jackson Immunoresearch, Goat antiArmenian hamster, adsorbed  to 
mouse Cat #127-065-160 and  BD Pharmingen has a cocktail to detect Armenian 
AND Golden Syrian  Cat #550335and this one will work nicely too.  Both are 
biotinylated and used diluted 1:250 or so.   If your antibody is NOT 
adsorbed against mouse, this may be a major source of your background.  If 
you already have an antiArmenian hamster secondary, you can do the 
adsorption yourself by diluting your secondary antibody in 1 or 2% mouse 
serum, let it stand for an hour or so and then apply it to the mouse 
spleen.  We prefer to buy the antibody already adsorbed and stay away from 
mouse serum with hamster antibodies, less work, reliable.

Our staining protocol:

Fix, block endogenous peroxidase (yours should work as long as you don't 
chew section off with higher conc of hydrogen peroxide).  I discussed this 

Normal serum block - yours looks fine, we go as high at 10% goat serum but 
we heat inactivate our normal serums to get rid of complement.

Do an Avidin/biotin block!!   Vector has a kit.

CD11c, your antibody concentration is in the ballpark, but we only incubate 
30 min at RT.  This antibody is diluted in 1% goat serum.

Secondary, goat antiArmenian Hamster, adsorbed to mouse, Jackson or BD 
Pharm cocktain diluted 1:250 or so, 30 min,.  This is diluted in the NORMAL 
SERUM BLOCK of  5% goat.

Strepavidin-HRP (TAGO/Biosource) diluted 1:500 (0.5 to 0.6 mg/ml original 
concentration) 20 min

Rinse with buffer containing only 0.05% Tween 20, and add 
chromogen.  Develop your POSITIVE control using a microscope, no exceptions 
in this lab.  We let a timer run to have approx time just for record 
keeping, an awareness of what to expect for future stainings.   We prefer 
AEC to DAB, better contrast.  We like Chris van der Loos's AEC, excellent!!

  Rinse off DAB, and proceed with hematoxylin counterstain, very light.  If 
you think your DAB is giving you trouble, try others,  DAKO DAB+ or 
Pierce  DAB  two part system or Pierce DAB enhanced for black ppt.  If the 
chromogen develops too fast, you may have to readjust the primary 
concentration.  Did you do a dilution panel to determine optimal working 
concentration of your primary?  If not, you may have it too concentrated 
for your other reagents, particularly since you are using DAB.   For 
dilution panel, work with 10 ug/ml and do dilutions from there.  You may be 
surprised at results and also eliminate background.

Good luck

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

   At 06:09 AM 10/7/2004, you wrote:
>I'm currently trying out to stain murine spleen and thymous for
>dendritic cells using the hamster anti-mouseCD11c antibody.
>As a secondary Ab I use biotin goat anti-hamster IgG and then
>streptavidin HRP for DAB staining. I'm getting a lot of background in
>both types of tissues even in the negative control. Below I have the
>protocol that I use.
>Frozen tissues cut at 10um placed on gelatin embedded slides.
>Air dry for 30min, fix in cold acetone for 10min and then in PBS/0.3%
>H2O2 for 10min for the endogenous peroxidase.
>Slides blocked with PBS/5% Goat serum for 30min.
>Primary Ab anti-mouse CD11c at 1:100 dilution for 1 hour.
>Secondary anti-hamster IgG at 1:800 dilution for 1 hour.
>Streptavidin HRP at 1:500 for 30min.
>Use DAB prepared solution 0.05% DAB- 0.015% H2O2 in 0.01M PBS pH 6.8
>The DAB I use is from Sigma.
>I'm not sure that the background is still from the endogenous
>peroxidases in the tissues or something else.
>I have tried staining the tissues with just the secondary Ab from 1:200
>up to 1:3200 dilution. In the high 1:3200 get very little background
>however I'm afraid that it might be too dilute to bind the primary Ab.
>Any suggestions would be greatly appreciated. Thanks
>Sotiris Tsalavos
>Institute of Immunology
>B.S.R.C. Alexander Fleming
>Athens, Greece
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