Eosin alcohol solution

Ovaries from all PND 23 female pups were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS at 4 °C. Samples were then dehydrated and embedded in paraffin according to standard procedures and cut into 5-μm serial sections. After deparaffinization, slides were stained with hematoxylin QS (Vector Laboratories, Burlingame, CA) for 30 sec, washed in running water, and stained with a 1% eosin/alcohol solution (Sigma-Aldrich) for 2 min. The stained slides were dehydrated and mounted with Poly-Mount (Polyscience Inc., Warrington, PA, USA). Images were captured with a Zeiss AxioVision microscope and software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) using the same exposure time for all images. Every fifth section was counted from the first section containing follicular cells until the last sections without follicular cells were observed. Only follicular cells that contained an oocyte with a nucleus were counted. Follicular cells were classified according to Pedersen and Peters (1968) . The small, medium and large follicular cells corresponded to type 1-3a, 3b-5a, and 5b-8 of their classification, respectively. In addition, only type 7-8 follicular cells were counted. The sections were counted twice, and the average of the two counts was calculated and reported.

Paraffin blocks of testis cultured tissue were prepared using standard
procedures; 5-μm serial sections were cut and mounted on slides.
After deparaffinization, sections were stained with hematoxylin QS (Vector Laboratories,
Burlingame, CA) for 30 s, washed under running water, and stained with a 1%
eosin/alcohol solution (Sigma-Aldrich, St. Louis, MO) for 2 min. The sections were
dehydrated and mounted with Poly-Mount (Polysciences, Warrington, PA). Images were
acquired with a Zeiss microscope and AxioVision software using the same exposure time for
all images.