Cellular localization of BM88 mRNA in paraffin-embedded rat brain sections by combined immunohistochemistry and non-radioactive in situ hybridization

• Published in: Brain research. Brain research protocols Vol. 7; no. 2; pp. 121 - 130
• Main Authors: Liang, Jing D, Liu, Jingang, McClelland, Phadungchom, Bergeron, Marcelle
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2001
• Subjects: Animals; Antibodies, Monoclonal; Antibody Specificity; Antisense Elements (Genetics); BM88; Brain; Brain Chemistry; Digoxigenin; Immunohistochemistry; Immunohistochemistry - methods; In situ hybridization; In Situ Hybridization - methods; Male; Membrane Proteins; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - immunology; NeuN; Neurons - chemistry; Paraffin Embedding; Rats; Rats, Sprague-Dawley; RNA, Messenger - analysis; Immunohistochemistry; Brain; BM88; NeuN; Cellular and molecular biology; Digoxigenin; In situ hybridization
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(01)00050-2

With the advent of gene cloning and sequencing, it has become increasingly common to identify novel genes for which no antibody is available. The best approach to study the expression and the distribution of these new genes is by in situ hybridization. One of the challenges with this method is to define the exact cellular subtype where the gene of interest is expressed. Conventional isotopic in situ hybridization methods lack precision for cellular identification because radioactive probes often result in a scattered signal. To identify the exact cellular subtype expressing BM88, we established a rapid colocalization method using non-isotopic in situ hybridization followed by chromogenic immunohistochemistry on the same tissue section. We demonstrated that BM88, which was identified from subtractive hybridization experiments between normal and ischemic tolerant brain tissue, was expressed exclusively in neurons in normal adult rat brain. Paraffin-embedded tissue was used as it resulted in better preservation of tissue and cellular morphology, thus allowing for more accurate histological localization of gene expression. It also allowed for retrospective studies on a number of archived tissue samples.