510 confocal microscopy

Mice were anesthetized with pentobarbital sodium and transcardially perfused with PBS containing 5 U/ml heparin. Brains were dissected, cryosectioned with a thickness of 14–18 mm, and fixed in ice-cold acetone. Sections were blocked with 5% normal goat serum for 1 h at room temperature and incubated overnight with primary antibody goat anti-CD13 (R&D Systems; AF2335; 1:200) which was diluted in blocking solution at 4 °C. Sections were washed in PBS and incubated with the secondary antibody Alexa 568-conjugated donkey anti-goat (Invitrogen; A11057; 1:200). Further, sections were stained with Dylight 488-conjugated tomato lectin (DL-1174, 1:100, Vector Laboratories) and coverslipped with fluorescent mounting medium (Dako, Carpinteria, CA, USA) to visualize brain microvessel with a 510 confocal microscopy (Zeiss). For thioflavin-S staining, brain sections were stained for 10 min with 0.2% thioflavin-S (T1892, Sigma-Aldrich) diluted in PBS. After repeated wash with PBS, brain sections were imaged using an IX53 fluorescence microscope (Olympus). The quantification of images was analyzed using the Image J software.

Testicular sections from 4 mice per group were analysed for stereological estimates and immunohistochemistry. Testes were fixed in Bouin’s fluid (Sigma Aldrich) or 10% Neutral buffered formalin (Fisher Scientific) at 4 °C overnight and treated through graded ethanol and Histoclear (National Diagnostics Ltd) ready for paraffin embedding. Serial sections (5–6 µm) were stained with a standard haematoxylin and eosin (H&E) and counting methods undertaken as previously described to quantitate spermatogenesis [26 (link)–29 (link)]. In addition, Immunofluorescence and Terminal deoxynucleotidyl transferase staining (TUNEL) staining (Roche, UK) performed with Zeiss 510 confocal microscopy for visualisation (see supplementary methods).