Immunohistochemical Approaches for the Study of Spermatogenesis

  • Cathryn A. Hogarth
  • Michael D. GriswoldEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 927)


Immunohistochemistry is an important technique that uses specific antibodies to determine the cellular localization of proteins/antigens in highly complex organs and tissues. While most immunohistochemistry experiments target protein epitopes, nonprotein antigens including BrdU may also be detected. Briefly, tissues are fixed, processed, sectioned, and then probed by a primary antibody while preserving the integrity of the tissue and cellular morphology. There are various methods available for visualization of the bound primary antibody that involve a reporter molecule which can be detected using light or fluorescent microscopy. Here we describe a basic immunohistochemistry protocol for identifying protein localization in testis sections using protein-specific antibodies.

Key words

Immunohistochemistry Testis Antigen retrieval Antibody Antigen Biotin Fluorophores 



The authors would like to thank Chris Small and Ryan Evanoff for their critical reading of the manuscript and Ryan for supplying the STRA8 immmunohistochemistry pictures. The work was supported by NIH grant HD RO1 10808 to MDG.


  1. 1.
    Coons A, Creech H, Jones R (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47:200–202Google Scholar
  2. 2.
    Ramos-Vara JA (2005) Technical aspects of immunohistochemistry. Vet Pathol 42:405–426PubMedCrossRefGoogle Scholar
  3. 3.
    Mighell AJ, Hume WJ, Robinson PA (1998) An overview of the complexities and subtleties of immunohistochemistry. Oral Dis 4:217–223PubMedCrossRefGoogle Scholar
  4. 4.
    Yamashita S (2007) Heat-induced antigen retrieval: mechanisms and application to histochemistry. Prog Histochem Cytochem 41:141–200PubMedCrossRefGoogle Scholar
  5. 5.
    Wilson DM III, Bianchi C (1999) Improved immunodetection of nuclear antigens after sodium dodecyl sulfate treatment of formaldehyde-fixed cells. J Histochem Cytochem 47:1095–1100PubMedCrossRefGoogle Scholar
  6. 6.
    Russell LD et al (1990) Histological and histopathological evaluation of the testis, 1st edn. Cache River Press, St. Louis, MOGoogle Scholar
  7. 7.
    Daneshtalab N, Dore JJ, Smeda JS (2010) Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies. J Pharmacol Toxicol Methods 61:127–135PubMedCrossRefGoogle Scholar
  8. 8.
    Miller RT, Groothuis CL (1990) Improved avidin-biotin immunoperoxidase method for terminal deoxyribonucleotidyl transferase and immunophenotypic characterization of blood cells. Am J Clin Pathol 93:670–674PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.School of Molecular Biosciences, Washington State UniversityPullmanUSA

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