Immunohistochemical differences between neurofilaments in perikarya, dendrites and axons: Immunofluorescence study with antisera raised to neurofilament polypeptides (200K, 150K, 70K) isolated by anion exchange chromatography

Abstract

Neurofilament (NF) proteins (70K, 150K and 200K D) were isolated from 2 M urea extracts of bovine spinal cord by anion exchange chromatography. Antisera to the individual NF polypeptides were produced in rabbits and affinity-purified on Sepharose columns prepared with their own antigen. The NF antisera were completely absorbed by their own antigen at protein concentrations that did not decrease the staining when the absorption was conducted with the heterologous NF antigens. Partial absorption (decrease in immunofluorescence titer) occurred at higher concentrations of the heterologous antigens. Cross-reactivity between the polypeptides of the NF triplet could not be detected by double immunodiffusion. The antisera formed immunoprecipitin lines only when reacted with their own antigen. Conversely, cross-reactivity was demonstrated by the immune blotting procedure. Anti-70K stained all three NF polypeptides. Anti-200K and anti-150K stained both 200K and 150K but not 70K, the main reaction being with their own antigen. The antisera were rendered monospecific by adsorption of the common antigenic determinants on Sepharose columns prepared with the heterologous NF antigens. The localization of the NF proteins was studied by immunofluorescence on cryostat sections of rat brain, cerebellum, spinal cord and posterior root ganglia. All NF antisera (anti-70K, anti-150K and anti-200K) stained axons including Purkinje cell baskets with identical pattern. Spinal cord motor neurons, posterior root ganglia neurons and pyramidal neurons in the cerebral cortex stained with anti-70K and anti-200K. No staining of neuronal perikarya and dendrites was observed with anti-150K. Aluminium-induced neurofibrillary tangles in rabbit spinal cord stained with anti-70K and anti-200K. The tangles were not decorated by anti-150K. It is concluded that a marked difference exists in the concentration of 150K depending on the location, i.e., cell body or axon; or, alternatively, that 150K undergoes modification of antigenic sites within the axon so that it may not be recognized immunologically as a component of the neurofilament within perikarya and dendrites.