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Proteomic analysis of immunostained, laser‐capture microdissected brain samples
Author(s) -
Moulédous Lionel,
Hunt Sybille,
Harcourt Rebecca,
Harry Jenny L.,
Williams Keith L.,
Gutstein Howard B.
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390026
Subject(s) - immunostaining , laser capture microdissection , proteomics , biology , immunofluorescence , microbiology and biotechnology , staining , antiserum , immunochemistry , gel electrophoresis , maldi imaging , pathology , microdissection , matrix assisted laser desorption/ionization , chemistry , antibody , immunohistochemistry , gene expression , biochemistry , medicine , immunology , genetics , organic chemistry , adsorption , desorption , gene
Proteomic analysis is often performed on homogenized preparations of whole tissues, which does not provide any information about relevant biochemical changes in specific cell types. Laser‐capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section. Phenotypically defined cells of interest may be visualized by immunostaining prior to microdissection. Previously published immunostaining protocols adapted to LCM require the use of very high antibody titers and very short incubation times. This raises the concern that low‐abundance antigens would not be detected and that antisera would be rapidly depleted. In addition, protein recovery from samples was not evaluated in most of these studies. Here, we describe an optimized immunostaining method based on immunofluorescence. By comparing two‐dimensional electrophoresis (2‐DE) results obtained from immunostained LCM brain tissue samples to those obtained from unstained, manually dissected samples, we demonstrated that immunofluorescent staining gave comparable protein recovery and similar resolution of protein spots on 2‐DE gels. Moreover, matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry analysis of selected spots from gels derived from control and immunostained LCM samples revealed that the immunostaining process had minimal effect on protein identification. LCM of immunofluorescently labeled tissue sections is a practical and powerful method to perform proteomic studies on specifically defined cell groups.