Biotin Tracer Permeability Assay for Blood-Testis Barrier

Permeability of the blood-testis-barrier to a biotin tracer was carried out as previously described^{49 (link)} with minor modifications. Briefly, EZ-link sulfo-NHS-LC-biotin (Thermo Scientific, UK) was freshly prepared as a 10 mg/mL solution in 0.01 M MgCl2 in PBS. 10–15 uL of biotin solution was injected into the testis under the tunica albuginea immediately after sacrifice, 10–15 uL of 0.01 M MgCl2 in PBS was injected into the contralateral testis as a control. Testes were left on ice for 30 mins and then fixed and processed as described above. The localisation of the biotin tracer was visualised by incubation with Streptavidin Alexa Fluor™ 546 conjugate (S11225, Thermo Scientific, UK), diluted 1:200 in PBS, for 1 hr at room temperature. Tissue sections were imaged using an LSM 780 confocal microscope (Carl Zeiss, UK).