Squash procedure for protein immunolocalization in meiotic cells.
Several techniques have been developed for protein immunolocalization in meiotic cells. However, most of them include treatments that lead to cell disruption and are only suitable for prophase-I cells. We describe a novel squash procedure of cell preparation for protein immunolabelling of different meiotic stages. This procedure is an alternative to both cryosectioning and whole spreading procedures. We present results obtained in mouse spermatocytes with three different antibodies: the MPM-2 mAb against mitotic phosphoepitopes, an anticentromere serum and a polyclonal serum against the SCP3 protein of the axial elements and lateral elements of the synaptonemal complex. The procedure was tested for single and double immunolabelling. With this technique a large number of cells at different meiotic stages can be analysed. Cell stages are easily identified and cell and chromosome structures are preserved. Thus, it allows the study of chromosome behaviour and the relationships between the different structural elements of the cell throughout meiotic divisions. Our procedure is also suitable for three-dimensional (3D) analyses and proved to be reliable in a wide range of systems including insects and mammals. In addition, the procedure may be interesting to obtain a rapid immunological diagnosis.
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Departamento de Biología, Universidad Autónoma de Madrid, Spain.
Suja
J A
JA
Santos
J L
JL
Rufas
J S
JS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018797:Cell Cycle Proteins; D002503:Centromere / Q000737:chemistry* / Q000648:ultrastructure; D002875:Chromosomes / Q000737:chemistry; D004268:DNA-Binding Proteins; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D008540:Meiosis; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000032:analysis*; D013090:Spermatocytes / Q000737:chemistry* / Q000166:cytology
D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C490062:Sycp3 protein, mouse