Brain, Testis, and Muscle Morphology Assessment

For the assessment of brain morphology and cAMP activation in testis, rats were transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4), followed by overnight post-fixation of the brains in the same fixative. For immunohistochemistry, rat brains were embedded in one gelatin block, and 40-μm coronal sections were freeze-cut and collected into 24 series (NeuroScience Associates, Knoxville, TN, USA). One of these series (containing 1 every 24 sections) was used for morphological analysis with anti-NeuN antibody (1:300, MAB377, Merck). Free-floating staining was performed as previously described [23 (link)]. Testes were collected and fixed as previously described [45 (link)]. Paraffin sections were stained with anti-cAMP (ab134902, abcam) at a dilution of 1:1000 and secondary goat anti-rabbit antibody (1:1000, BA-1000, Vector Laboratories). For muscle staining, rat gastrocnemii were rapidly frozen in liquid nitrogen directly after dissection, then stored at −80 °C till sectioning. Cross-sections with 8-µm thickness were stained using a Trichrome Stain Kit (ab150686, Abcam).