[A fast method for preparing the mouse germ cell slides].
OBJECTIVE
To establish a new method of fast preparation of mouse germ cell slides for immunofluorescence assay.


METHODS
Mice of different developing stages (at postnatal days 16, 21, 28 and 120) were treated with colchicine by intraperitoneal injection respectively, and 3 hours later, the testes were taken out and the tunica albuginea were removed. The seminiferous tubules were separated and minced into small pieces, resuspended with ice-cold PBS, and the cells were filtered and collected by centrifugation. After washed with ice-cold PBS for three times, the cells were treated with a hypotonic solution, fixed with 4% paraformaldehyde, and finally mounted onto the slides. To test the slide quality, the cells mounted on the slides were stained with DAPI, and immunofluorescence staining was performed using 21-day-old mouse slides.


RESULTS
Stained with DAPI, the distribution of various germ cells was observed on each slide by the new method and the background was clear. According to the size and color of the cell nucleus, the types of germ cells could be recognized preliminarily. The types of germ cells on the slides were very different among 16-, 21-, 28- and 120-day-old mice, of which the slides from 28-day-old mice had the most types of germ cells. Immunofluorescence staining using 21-day-old mice slides revealed that the red- and green-fluorescence labeled target proteins were observed in spermatocytes, and the background was clear, suggesting the slides were suitable for immunofluorescence detection.


CONCLUSIONS
A new method of fast preparing the mouse germ cell slides was successfully established, providing a foundation for the study on the process of spermatogenesis.
Department of Pharmacy, Third Xiangya Hospital; School of Pharmaceutical Science, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Meng
M
Yang
Department of Pharmacy, Third Xiangya Hospital; School of Pharmaceutical Science, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Xiaocong
X
Zuo
Center for Experimental Medicine Research, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Yan
Y
Li
Center for Experimental Medicine Research, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Lihua
L
Huang
Center for Experimental Medicine Research, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Xiaowei
X
Xing
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005786:Gene Expression Regulation; D007211:Indoles / Q000378:metabolism; D034921:Lamin Type B / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D013091:Spermatogenesis; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism; D013194:Staining and Labeling; D013737:Testis / Q000166:cytology; D013997:Time Factors
D007211:Indoles; D034921:Lamin Type B; D008565:Membrane Proteins; C559752:SUN5 protein, mouse; C007293:DAPI