Laminin and BrdU co-staining protocol, anybody?

One friend of mind is looking for a proper double staining protocol for laminin and BrdU on fixed rat striatal muscle slices. Can anybody help, please?

The following antibodies are used:

Primary antibodies:

Purified Anti-BrdU Mouse Cat No.: 555627 BD Pharmingen

Anti-Laminin, antibody produced in rabbit Cat No.: L9393 Sigma

Secondary antibodies:

Alexa Fluor 488 goat anti-mouse IgG Cat No.: A11001 Invitrogen

Alexa Fluor 546 goat anti-rabbit IgG (H+L) Cat No.: A11010

5 thoughts on “Laminin and BrdU co-staining protocol, anybody?

  1. Atilla,

    I just so happens that we have a company here who does this kind of work HSRL: http://www.hsrl.org/

    They say that you need to use OCT Compound (available through Fisher) to avoid interference with flour antibodies and have a means of making the slices. They are also willing to give them a quote to do it.

    If you search PubMed/Google Scholar for OCT compound and laminin, you’ll find a number of references for protocols.

  2. Hey Attila,

    It’s been a while since I’ve commented over here.

    I would recommend your colleague use light microscopy in place of fluorescence. Use a different BrdU antibody: The Roche Anti-BrdU POD (peroxide dismutase) at 1:40 is easily developed using a DAB developer.

    The laminin antibody can then be developed using a biotinylated anti-rabbit that could be developed using any number of the Vector kits. (I’d recommend Vector red for muscle)

    Then counterstain with hematoxylin, and voila, you’ve got red muscle cells, brown dividing nuclei and blue nuclei elsewhere…

    Email me if you want more specifics. I know many people think fluorescence is the best type of microscopy, but I have found that muscle histology is better done with regular chromogenic methods. It’s what the medical pathologists use, and its much more rugged than delicate fluorophores.

    Good luck to your friend.

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