Cy3 conjugated anti sheep

Sperm was stained using sheep polyclonal antibodies against Np detecting Np65 and Np55 (pan-Np55/65 1:500, R&D systems). Secondary antibodies were Cy3-conjugated anti-sheep (1:1000, Jackson Immunoresearch). Counterstaining used phalloidin-iFluor 488 green (1:1000, Abcam, Berlin, Germany) and fluoromount g DAPI (Southern Biotech). Immunofluorescence was visualized using a Leica SP5 confocal microscope.

Paraffin-embedded testis sections were deparaffinized and blocked with BSA (5% in phosphate-buffered saline) for 1 h and then incubated with primary antibodies (sheep polyclonal Np detecting Np65 and Np55 (pan-Np55/65; 1:500, R&D systems) overnight at 4 °C in a humidified chamber. Post incubation, the sections were washed with PBS and probed with the secondary antibody (Cy3-conjugated anti-sheep 1:1000, Jackson Immunoresearch) in a sequential manner. Sections were examined by an Axioplan2 (Zeiss), and images were captured by Spot RT-KE camera (Diagnostic Instruments, Marburg, Germany).

Adult mice were anesthetized with isoflurane and transcardially perfused with PBS, followed by 4% PFA. Testes were dissected and post-fixed in 4% PFA overnight. Testis sections were blocked (20% horse serum in PBS, one hour, room temperature) and incubated with primary antibodies in PBS containing 0.3% Triton X-100 and 10% horse serum (overnight, 4 °C). Primary antibodies used were sheep polyclonal anti-Np detecting Np65 and Np55 (pan-Np55/65; 1:300, R&D systems) and rabbit polyclonal anti-Stra8 (Stimulated By Retinoic Acid 8, used as a marker for spermatogonia and spermatocytes, 1:500, Abcam), anti-CPY11A1 (Cytochrome P450 Family 11 Subfamily A Member 1, marker for Leydig cells, 1:500, Abcam), anti-alpha-smooth muscle actin (marker for myoid cells, 1:500, Abcam), and anti-Sox9 (SRY-Box Transcription Factor 9, marker for Sertoli cells, 1:500, EMD Millipore, Darmstadt, Germany) antibodies. The specificity of antibodies was assessed with negative controls using pre-immune sera.
Secondary antibodies were Cy3-conjugated anti-sheep and Cy5-conjugated anti-rabbit (1:1000, Jackson Immunoresearch). After washing with PBS and briefly with water, the sections were mounted on glass slides with fluoromount g DAPI (Southern Biotech, Birmingham, AL, USA) and visualized using a Leica SP5 confocal microscope (Leica, Wetzlar, Germany).