Immunohistochemical Localization of Adhesion Molecules - Adhesion Molecules - page 25

control. Next is applied a secondary antibody against the primary antibody. h e
secondary antibody has a biotin molecule attached to it or is said to be biotinylated.
h e slides are then incubated in a conjugated streptavid i n horseradish peroxidase
solution. As discussed above, avidin is a glycoprotein that has a very strong ai nity
for the protein biotin, so there is a strong binding reaction between the streptavidin
and the biotinylated secondary antibody. h e i nal reactive step is to add a visible
marker or dye. h e most frequently used dye is 3,3¢-diaminobenzidine, a visible
dye, that binds to streptavidin and in the presence of peroxidase produces a brown
precipitate ( Fig. 3 ) . h ere are other chromogens that yield red, purple, or blue i nal
reaction colors, but brown is the most commonly used. h e ability to use more
than one color is very helpful for co-localizing dif erent proteins in the same tissue
or cell as in Fig. 4 (Schmetz et al. 2001). It is also possible to attach l uorescent
dyes to the secondary antibody to produce brilliant colors under a l uorescent
microscope ( Fig. 5 ) or a confocal microscope ( Fig. 4 ).
Fig. 3 Immunohistochemistry of p120 in MLO-Y4 osteocytes using a 3,3¢-diaminobenzidine
marker dye. Immunohistochemistry using monoclonal anti-mouse antibodies against p120 in
MLO-Y4 cells. Let , a control with no primary antibody. Right, with a primary antibody and
3, 3¢-diaminobenzidine marker dye. Original = 400 × (MLO-Y4 cells were kindly provided by Dr.
Lynda Bonewald, Dept. Oral Biology, U. Missouri at KC School of Dentistry).
Color image of this i gure appears in the color plate section at the end of the topic.
Early Embryology
Development and dif erentiation of multicellular organisms is critically dependent
on cell-cell adhesions (Trejo et al. 2008). Early 1-4-cell embryos mark the change