Question.

I have been trying to stain for microglia in paraffin sections of rat brain using peroxidase-labeled Griffonia simplicifolia lectin (GSI-B4-HRP) from Sigma.  It has been used in various papers for staining of active and resting microglia but  I cannot seem to get it to work. Are there any tricks that I might be missing?

Answer.

I have not used this lectin for microglia but have used it for other things. The purity varies considerably because the seeds of Griffonia, when extracted, may yield just one lectin or several isolectins (depending on the seeds), and the B4 lectin is then purified from this mixture. I have found a lot of variation from batch to batch but more so from manufacturer to manufacturer. The best luck I had with this lectin was from Vector Laboratories, Burlingame, California, who specialize in the production of lectins. I have also had problems with some lectin-HRP conjugates. In my experience the conjugates (especially the HRP ones) have only a limited shelf life and this can lead to background staining. Part of your problem may be that lectin binding can be significantly altered by fixation and processing. I would suggest that you first try it on frozen sections to determine whether the conjugate you have is working.

This lectin usually requires the availability of calcium ions to bind. If you are using OCT freezing compound, this contains sufficient calcium if you don't remove the OCT before staining.

I do not have the latest Vector catalog available at the moment but believe that they have an antibody against GSI B4. This might be a better approach if the problem is one of conjugate breakdown or excessive background staining.

Another point is that the lectin binding can be easily confirmed with negative (inhibited) controls, inhibitors for GSI B4 include:

        o,p-Nitrophenyl-N-acetyl-alpha-galactosamine
        Galactose-N-acetyl-alpha-1,3-galactose
        Methyl-N-acetyl-alpha-galactosamine
        N-acetylgalactosamine

Barry R. J. Rittman
Univ. Texas HSC Dental Branch
Houston, Texas
(brittman[AT]mail.db.uth.tmc.edu)