A new approach for optimal morphological identification and immunolabeling of spermatogonial cells.
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.
1Laboratório de Biologia Estrutural e Reprodução,Departamento de Morfologia,Universidade Federal de Minas Gerais,31.270-901 Belo Horizonte,Brasil.
Amanda V
AV
Albuquerque
1Laboratório de Biologia Estrutural e Reprodução,Departamento de Morfologia,Universidade Federal de Minas Gerais,31.270-901 Belo Horizonte,Brasil.
Ana Luiza
AL
Drumond
2Department of Experimental Radiation Oncology,MD Anderson Cancer Center,University of Texas,77030 Houston,USA.
Shan
S
Shao
3Laboratório de Biologia Celular,Departamento de Biologia,Universidade Federal de Juiz de Fora. 36.036-900 - Juiz de Fora,Brasil.
Rossana C N
RC
Melo
1Laboratório de Biologia Estrutural e Reprodução,Departamento de Morfologia,Universidade Federal de Minas Gerais,31.270-901 Belo Horizonte,Brasil.
Fernanda R C L
FR
Almeida
2Department of Experimental Radiation Oncology,MD Anderson Cancer Center,University of Texas,77030 Houston,USA.
Marvin L
ML
Meistrich
1Laboratório de Biologia Estrutural e Reprodução,Departamento de Morfologia,Universidade Federal de Minas Gerais,31.270-901 Belo Horizonte,Brasil.
Hélio
H
Chiarini-Garcia
D000818:Animals; D008297:Male; D008810:Mice, Inbred C57BL; D013093:Spermatogonia / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods*; D013737:Testis / Q000166:cytology*; D016610:Tissue Embedding / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*