Biomedical Engineering Reference
In-Depth Information
Fig. 2
Focal contacts demonstrated by TIRF. Focal contacts are specialized at achments between
integrins and actin i laments that allow cells to pull on the substratum to which they are at ached.
h is i gure was taken using Total Internal Rel ection Fluorescence (TIRF) imaging of a smooth
muscle cell in culture using the antibody p-Tyr-FITC, Sigma #F3145 (clone #PT66). Courtesy of
Soon-Mi Lim and Andreea Trache, Texas A&M Health Science Center College of Medicine.
Color image of this i gure appears in the color plate section at the end of the topic.
reaction must be blocked. h e i rst of these reactions is a block against enzymatic
endogenous peroxidase activity with a solution of hydrogen peroxide and sodium
azide. h is is very important because the step (see below) that attaches the visible
dye reacts with peroxidase to produce a brown precipitate and it is critical that it
react with only the intended molecule. Next is a casein and protein mixture, called
a background Sniper, which is required to reduce nonspecii c protein background
staining. h is is followed by an avidin-biotin blocker to suppress endogenous biotin.
Avidin is a glycoprotein that has a very strong ai nity for the protein biotin. h is
is important because the secondary antibody that is going to be applied later has a
biotin molecule attached to it and must be the only biotin molecule 'visible' to the
dye that will be used to color the reactive tissue. Once the tissue is prepared with all
extraneous proteins blocked so that only the protein of choice remains available,
the tissue is rinsed with buf er and the appropriate primary antibody is applied.
h is antibody will react with only the specii c antigen to which it is antagonistic.
It is important to omit the primary antibody step in some of the tissue as a negative
