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A New Approach for Optimal Morphological Identification and Immunolabeling of Spermatogonial Cells

Published online by Cambridge University Press:  16 May 2014

Amanda V. Albuquerque ,
Ana Luiza Drumond ,
Shan Shao ,
Rossana C.N. Melo ,
Fernanda R.C.L. Almeida ,
Marvin L. Meistrich  and
Hélio Chiarini-Garcia
Amanda V. Albuquerque
Affiliation:
Laboratório de Biologia Estrutural e Reprodução, Departamento de Morfologia, Universidade Federal de Minas Gerais, 31.270-901 Belo Horizonte, Brasil
Ana Luiza Drumond
Affiliation:
Laboratório de Biologia Estrutural e Reprodução, Departamento de Morfologia, Universidade Federal de Minas Gerais, 31.270-901 Belo Horizonte, Brasil
Shan Shao
Affiliation:
Department of Experimental Radiation Oncology, MD Anderson Cancer Center, University of Texas, 77030 Houston, USA
Rossana C.N. Melo
Affiliation:
Laboratório de Biologia Celular, Departamento de Biologia, Universidade Federal de Juiz de Fora. 36.036-900 - Juiz de Fora, Brasil
Fernanda R.C.L. Almeida
Affiliation:
Laboratório de Biologia Estrutural e Reprodução, Departamento de Morfologia, Universidade Federal de Minas Gerais, 31.270-901 Belo Horizonte, Brasil
Marvin L. Meistrich
Affiliation:
Department of Experimental Radiation Oncology, MD Anderson Cancer Center, University of Texas, 77030 Houston, USA
Hélio Chiarini-Garcia*
Affiliation:
Laboratório de Biologia Estrutural e Reprodução, Departamento de Morfologia, Universidade Federal de Minas Gerais, 31.270-901 Belo Horizonte, Brasil
*
*Corresponding author. [email protected]; [email protected]
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Abstract

High quality fixation often inactivates epitopes and gentler fixation can fail to preserve biological structure at the required resolution. For studies of male reproduction, immunofluorescence techniques using paraformaldehyde fixation associated with paraffin as an embedding medium gives good epitope preservation, although the cell becomes morphologically compromised. On the other hand, glutaraldehyde associated with a plastic resin has been used with success to recognize and distinguish each spermatogonial cell subtype, but the antigenic sites become inaccessible to antibodies. Here we describe a new method that provides excellent morphological details of testicular cells while preserving the binding capacity of epitopes. Using a combination of glutaraldehyde and paraformaldehyde as a fixative and LR White resin for embedding, we show that it is possible to clearly recognize spermatogonial subtypes (Aund, A–A4, In and B spermatogonia) on 1-μm thick-sections and to label epitopes such as bromodeoxyuridine, a marker used for cellular cycle studies in the testis. The information gained from this procedure can be critical for understanding spermatogonial process of self-renewal and differentiation.

Keywords

spermatogoniaLR White resinhigh-resolution light microscopyimmunofluorescenceplastic resin embedding
Type
Techniques and Instrumentation Development
Information
Microscopy and Microanalysis , Volume 20 , Issue 4 , August 2014 , pp. 1304 - 1311
Copyright
© Microscopy Society of America 2014 

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