Lsm780 laser scanning microscope

Testes and ovaries were fixed in 10% buffered formalin, embedded in paraffin, and sectioned to examine using standard hematoxylin/eosin counterstain. Images were processed using Adobe Photoshop software. Specifically, background was whitened, brightness and contrast were adjusted, and the sharpening edges filter was applied. Slides were deparaffinized in xylene and rehydrated in a descending alcohol gradient. Antigen retrieval was performed using 10 mM citrate buffer (pH 6.0) for 10 min. Sections were allowed to cool down to room temperature and washed in PBS. To quench endogenous fluorescence, slides were incubated in a solution of 0.1% Sudan Black in 70% ethanol for 15 min, washed in PBS, and blocked with 10% horse serum in PBS for 1 h at room temperature. Primary antibodies were diluted as described above and incubated overnight at 4°C. The following day, slides were washed in PBS, then incubated for 1 h at room temperature with a host-specific secondary antibody conjugated with Alexa fluor dyes (Invitrogen Life Technologies and Bethyl), and counterstained with blue fluorescent 4′,6-diamidino-2-phenylindole (DAPI). Images (Z-stacks with 0.6-µm optical sections, 16 sections total) were acquired using a Zeiss LSM780 laser scanning microscope. Maximum intensity projections were generated using ImageJ software.