Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.
Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.
Riederer
B M
BM
Institut d'Anatomie, Université de Lausanne, Switzerland.
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D001923:Brain Chemistry; D002415:Cats; D002531:Cerebellum / Q000033:anatomy & histology / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D016900:Neurofilament Proteins / Q000276:immunology*; D013116:Spinal Cord / Q000033:anatomy & histology / Q000276:immunology; D016707:Tissue Fixation
D000447:Aldehydes; D000906:Antibodies; D016900:Neurofilament Proteins