RE: Fluorescent immunohistochemistry

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From:Abizar Lakdawalla <>
To:"'Johnson, Jennifer(Hist)'" <>,
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Hi Jennifer, here is an outline of the procedure I would recommend:
1. Dewax slides (if paraffin embedded)
2. Block with Power Block (or equivalent kappa casein based blocking
reagent; use of serum blocking reagents increases fluorescence background)
3. Incubate with primary antibody (the rabbit polyclonal would be a better
first choice). Wash.
4. Incubate with biotinylated anti-rabbit secondary antibody. Wash.
5. Incubate with streptavidin-phosphatase. Wash.
6. Incubate with Fast Red chromogen (the fast red precipitate is insoluble
in water and is brightly fluorescent - works with texas red filters). Wash.
7. Place the slides in Retrievit-6 solution and microwave till solution
reaches approx. 70C. About 30 sec for 5 slides). Wash. This step removes the
antibodies and enzyme conjugates without affecting the fast red precipitate.
8. Block with PowerBlock again.
10. Incubate with the next primary antibody (the anti-CD68). Wash.
11. Incubate with biotinylated anti-mouse secondary antibody. Wash.
12. Incubate with Streptavidin conjugated to Fluor-Green (or any other green
fluorescent dye like one of the Alexa series). Wash.
13. Incubate with DAPI counterstain. Wash.
14. Mount in Supermount.
15. Examine with appropriate filter sets. The drug should show up as
orange-red, the CD68 as green, and the nuclei will be a bright blue.

Will fax you the relevant manuals if you send me your fax number.


-----Original Message-----
From: Johnson, Jennifer(Hist) []
Sent: Thursday, April 13, 2000 11:46 AM
To: 'Abizar Lakdawalla'
Subject: RE: Fluorescent immunohistochemistry

Thank you for your reply to my histonet query.
Here is the information that you requested.
1. and 2.  One antibody is Mouse anti-human CD 68 and the other is rabbit
anti-human for a drug we designed.

3. I have a texas red, a dapi and a flourescein filter set.

4.  the Mouse anti CD 68 is in a higher concentration in the tissues than
the rabbit anti-drug.
I would appreciate any info you could give me on your products.  Thanks,
Jennifer Johnson

-----Original Message-----
From: Abizar Lakdawalla []
Sent: Thursday, April 06, 2000 2:09 PM
To: Johnson, Jennifer(Hist);
Subject: RE: Fluorescent immunohistochemistry

We offer fluorescent kits which are based on the streptavidin-biotin
interaction so the procedures should integrate well with your current set
up. I would like to get some more information from you before I suggest a
1. What is the antibody species (mouse, rabbit, etc) for the antibody
against one of the antigens?
2. What is the second antigen antibody species?
3. What kind of filter sets you have for your fluorescence microscope?
4. Which antigen is present in greater concentration?


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