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

Details

Serval ID
serval:BIB_212BABC18FD1
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.
Journal
Brain Research Bulletin
Author(s)
Riederer B.M.
ISSN
0361-9230 (Print)
ISSN-L
0361-9230
Publication state
Published
Issued date
1993
Volume
30
Number
5-6
Pages
623-627
Language
english
Abstract
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.
Keywords
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
Create date
24/01/2008 14:34
Last modification date
20/08/2019 12:57
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