Re: hamster & mouse on mouse (long answer)

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From:Abizar Lakdawalla <>
To:kwalters <>
Content-Type:text/plain; charset=us-ascii

Hi Kathy, this is how we do similar projects in house (paraphrased from our
Do an iso-IHC procedure with the mouse primary antibody, streptavidin-HRP, and
DAB; treat DAB stained slides with "Retrievit-6": do another IHC staining with
the hamster antibody, anti-hamster secondary antibody (adsorbed with mouse
serum or mouse acetone powders), streptavidin-HRP, AEC, hematoxylin, and
mounting medium.

Note: For a double staining you have to do a minimum of 4 slides, 1 for
negative control, 2 for staining only with antibody A, 3 for staining only with
antibody B, and 4 for staining with antibody A and antibody B. Below is the
double staining procedure for Slide 4.

Step 1: Prelabel the mouse primary antibody with 1/10th volume of the
"biotinylation reagent" (this adds biotin residues to the primary antibody).
Step 2: Prepare the tissue sections by dewaxing, peroxide block (5 min, wash),
avidin-biotin block (10min, wash, 10min, wash), and "PowerBlock" (3min).
Step 3: Add 1/10th vol of "MouseBlock" reagent to the prelabeled primary
antibody (from step 1), incubate for about 5 minutes and place the mixture on
the mouse tissue section. Incubate for about 30 minutes (at RT). Wash the
unbound antibody off with PBS-Tween-20.
Step 4: Add Streptavidin-HRP (20min/RT, wash) followed by DAB chromogen (about
10min). Wash in running water (2min).
Step 5: Cover slide with Retrievit-6 and steam for 2-5 minutes (or in microwave
for about 45 seconds), wash in water (2min).
Step 6: Block slides again with PowerBlock (3min, drain well). Then add the
Hamster antibody and incubate for the required time. Wash with PBS-Tween-20.
Step 7: Add mouse adsorbed anti-hamster antibody (biotinylated), incubate for
20 min. Wash in PBS-Tween-20.
Step 8: Add Streptavidin-HRP (20min/RT, wash) followed by AEC chromogen (about
10min). Wash in running water (2min). Counterstain with hematoxylin. Wash and

Brown staining will indicate the presence of the antigen detected by the mouse
antibody, red staining will indicate the antigen detected by the hamster
antibody, and the nuclei will be stained blue.


> Hello oh wise ones,
> I wonder if anyone can advise me on a project I need to start.  I have to
> double label mouse spleen and thymus with antibodies (one from hamster; one
> from mouse).  Are these species as close as I imagine?
> Kathy

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