Differential immunostaining for substance P in Huntington's Diseased and normal spinal cord: significance of serial (optimal, supra-optimal and end-point) dilutions of primary anti-serum in comparing biological specimens

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Abstract
Using immunocytochemistry (ICC), the number of immunoreaction products (IRPs) visualized for the peptide substance P (SP) appears reduced within the human spinal cord (also substantia nigra) taken at autopsy from cases diagnosed with Huntington's disease (HD) compared with the non-HD cases (Vacca 1983). The reductions of SR-IRPs become apparent in the HD specimens when primary anti-SP serum is applied to the tissue sections at “supra-optimal” dilutions; that is, dilutions greater than the “optimal” dilutions which visualize maximal numbers of SP-IRPs and concomitantly give maximal staining intensity. Curiously, the application of “optimal” dilutions to the HD and non-HD specimens visualizes equivalent numbers of SP-IRPs; therefore, the qualitative and quantitative differences between the specimens become masked. Applying “supra-optimal” dilutions of the anti-SP serum unmasks the difference, and also reveals different end-points for the immunostain deposited in each type of specimen. In both the HD and non-HD specimens, two sizes of SP-IRPs could be identified, large (3 μm) and small (0.7 μm). Presumably they mark two different categories of axons defined by caliber (e.g.; C-type and A γ), or origin (e.g., sensory intrinsic, or supraspinal). Alternatively the large SP-IRPs label tangentially-cut or large axons and nerve terminals. In the present report, counts of the large and small SP-IRPs visualized in the HD and the non-HD specimens have been plotted against the serial dilutions (optimal, supra-optimal and endpoint) of the primary anti-SP serum used for ICC. The graphs which result, describe a “titration curve” characteristic for the large SP-IRPs in each specimen. Applying supra-optimal dilutions of the primary antibody, causes the numbers of large and small SP-IRPs to decrease differentially. The numerical decrease probably largely reflects the concentration of immunoreactive antigen in different anatomic or metabolic tissue compartments. Alternatively, or perhaps to a lesser degree, the small and large SP-IRPs represent cross-reacting components which have different staining end-points, and may have biological significance in HD. Criteria are given for the proper use of ICC, and have significance regarding the use of commercial kits especially for pathological diagnosis.

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