Immunohistochemical Protein Quantitation: Establishing Optimal Conditions For Linear Changes In Signal Detection As A Function Of Bound Antibody And The Relationships Be-Tween Staining Intensity, Protein Levels, And Messenger Rna For Tyrosine Hydroxylase Within Rat Brain Tissue Sections
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Type of WorkText
ProgramDoctor of Philosophy
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Immunohistochemistry (IHC) is used in pathology for diagnosis of dis-ease and in basic research for studying where biomolecules reside. It is prob-lematic, however, that quantitative evaluation of substances stained by IHC is not well accepted. Demonstration that product formed by an antibody/enzyme complex (i.e., stain) is linear with respect to the amount of antibody bound is key. The objective of this work was devise assay conditions that enable a linear relation between the stain level and amount of protein within tissue through ap-plication of the commonly used avidin-biotin complex (ABC) immunoperoxidase staining to freely floating fixed brain sections. We hypothesized that if sufficient time for reactants to bind is employed, staining will reflect the number of primary antibody molecules bound to the antigen and thus generate a linear staining pattern as the primary antibody is systematically reduced. If the antigen levels change, a shift in the staining will occur and the magnitude of shift will mirror the magnitude of antigen change. Using detection of tyrosine hydroxylase (TH), an enzyme found in noradrenergic, dopaminergic, and adrenergic neurons, and examining two cell populations with very different levels of enzyme expression or applying an experimental but physiologically relevant manipulation known to affect the enzyme's synthesis, we demonstrate that consistently higher anti-TH concentrations are required when TH expression is reduced, and that times of incubation between 48 and 72 h provide maximal ability to discern weak from strong staining. Differences (reductions) of TH staining intensity of 7- to 8-fold required 10-fold higher antibody concentrations to achieve equal staining; and conditions where intensity was reduced by 3.5- to 4-fold required 3-fold higher antibody concentrations. Our studies showed also that short anti-TH incubation times (< 48 h) reduced the sensitivity of assay. Very short times (< 1 h) produced no linearity of response but rather, it was all-or-none. Finally, we used 4 differ-ent anti-TH antibodies and determined that optimum staining was dependent on the amount of immunoglobulin G (IgG) contained in the antiserum. All achieved identical staining when the IgG levels were normalized.