Goat anti mouse cf 568

For IF staining, testis sections, and sperms were permeabilized with PBS pH 7.4 containing 0.1% Triton‐X‐100 for 30 min. Antigen retrieval was performed by putting slides in a pressure cooker for 3 min in 0.01 M citrate buffer (pH 6.0). Non‐specific binding sites were blocked with PBS containing 5% BSA and normal goat serum diluted 1:5.³⁴ Sections were incubated with primary antibodies anti‐RSPH6A (1:100); anti‐SYCP3 (1:100; #sc‐74 569; Santa Cruz Biotechnology), and anti‐α‐Tubulin (1:100) at 4°C. After three washes in PBS, slides were incubated for 1 h with PNA lectin (#L32458; Thermo Fisher Scientific) diluted at 1:50, and the appropriate secondary antibody [goat anti‐rabbit Alexa Fluor 488, (#A32731 Thermo Fisher Scientific); goat anti‐mouse CF™ 568 (#SAB4600082; Sigma–Aldrich)] both diluted to 1:500 in the blocking mixture for 1 h at RT. Nuclei were stained with Vectashield + DAPI and slides were observed and captured with the optical microscope (Leica DM 5000 B + CTR 5000) with a UV lamp and saved with IM 1000 software. Each IF was performed in triplicate.

The SPZ were fixed in 4% paraformaldehyde in PBS for 10 min at RT and then in PBS and incubated with 0.1% Triton X-100 (#T8787; Sigma-Aldrich, Milan, Italy) in PBS [76 (link)]. The slides were blocked with a PBS solution containing 20% of normal goat serum (#NS02L Sigma-Aldrich, Milan, Italy) and 5% BSA (#05470; Sigma-Aldrich, Milan, Italy) before the addition of the abovementioned primary antibodies and anti-α-tubulin antibody (#E-AB-20036; Elabscience Biotechnology, Wuhan, China) all were diluted to 1:100 and incubated overnight at 4 °C. After washing with PBS, the slides were incubated for 1 h with PNA lectin (#L32458; Thermo Fisher Scientific, Waltham, MA, USA) diluted at 1:50, and the appropriate secondary antibody [goat anti-rabbit Alexa Fluor 488, (#A32731 Thermo Fisher Scientific, Waltham, MA, USA); goat anti-mouse CF™ 568 (#SAB4600082; Sigma–Aldrich, Milan, Italy)] both diluted to 1:500 in the blocking mixture. In all IF analyses, the slides were mounted with DAPI for nuclear staining and observed under a fluorescent microscope.

The P1a and P1b antibodies recognize the NimC1 molecule on plasmatocytes, the L1a, L1b and L1c antibodies [36 (link)] react with the Atilla molecule on lamellocytes [37 (link), 38 (link)]. The antibodies were used as neat in the form of hybridoma tissue culture supernatants. The secondary antibody was goat anti-mouse CF-568 (Sigma-Aldrich), used at 1:1000 dilution.