Streptavidin alexa fluor 546 conjugate

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).

Primary antibodies are: rabbit anti-mouse MBP (Dako A0623) 1:1000; rabbit anti-mouse GST-π (MBL 312) 1:500; Rabbit anti-Iba1 (Wako 019–19741, 50 μg/100 μl) 1:500; rabbit anti-GFAP (Dako Z0334) 1:5000; rabbit anti-mouse MOG (abcam ab32760) 1:100; rabbit anti-dMBP (Millipore AB5864) 1:3000; mouse anti-Neurofilament (Covance SMI312) 1:5000; rabbit anti-mouse ALDH1L1 (abcam ab87117) 1:1000; rabbit anti-mouse NeuN (Millipore ABN78) 1:2000; rat anti-mouse CD107a (Biorad MCA4707T) 1:200; rabbit anti-mouse NG2 (abcam ab129051) 1:200; rabbit anti-mouse ki67 (abcam ab16667, clone SP6) 1:100; goat anti-Iba1 (Thermo Fisher Scientific PA5–18039) 1:250.
Secondary detection antibodies are: Goat anti-rabbit IgG biotinylated (Jackson ImmunoResearch 111–065-144) 1:1000; Goat anti-rabbit IgG biotinylated (Vector BA-1000) 1:200 or 1:1000; Goat anti-mouse IgG biotinylated (Vector BA-9200) 1:1000; Goat anti-rat biotinylated (Vector BA-9400) 1:200; donkey anti-rabbit IgG (Jackson ImmunoResearch 711–065-152) 1:200; Streptavidin, Alexa Fluor™ 546 conjugate (Thermo Fisher Scientific S11225) 1:800; donkey anti-goat IgG Alexa Fluor™ 488 (Thermo Fisher Scientific A11055) 1:500.