Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Détails

ID Serval
serval:BIB_212BABC18FD1
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.
Périodique
Brain Research Bulletin
Auteur(s)
Riederer B.M.
ISSN
0361-9230 (Print)
ISSN-L
0361-9230
Statut éditorial
Publié
Date de publication
1993
Volume
30
Numéro
5-6
Pages
623-627
Langue
anglais
Résumé
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.
Mots-clé
Aldehydes/pharmacology, Animals, Antibodies/immunology, Antibodies/isolation & purification, Blotting, Western, Brain Chemistry, Cats, Cerebellum/anatomy & histology, Cerebellum/immunology, Electrophoresis, Polyacrylamide Gel, Immunohistochemistry, Neurofilament Proteins/immunology, Spinal Cord/anatomy & histology, Spinal Cord/immunology, Tissue Fixation
Pubmed
Web of science
Création de la notice
24/01/2008 14:34
Dernière modification de la notice
20/08/2019 12:57
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