Alexa fluor 488 affinipure goat anti mouse igg

For immunofluorescence assay of tissue sections, deparaffinizated sections described above were washed in phosphate-buffer saline (PBS) for 5 min. These sections were boiled in sodium citrate buffer for antigen retrieval for 15 min, and blocked in 5% BSA for 45 min. For immunofluorescence of spermatozoa, slides were treated with 3-Aminopropyl (APES, Sigma, 919-30-2) in advance. 50 μl of sperms at 1 × 10⁶/ml was added onto the APES treated slides. Slides were fixed in 4% PFA, and blocked in 5% BSA supplemented with 0.3% Triton X-100 for 30 min. Slides were incubated with primary antibody, anti-SEPTIN12 (1:100, Abnova, H00124404-M), or anti-PLCζ (1:100, Abcam, ab124446), overnight at 4°C, and then detected by Alexa Fluor 488-AffiniPure Goat Anti-Mouse IgG (Jackson, 115-545-003) or Alexa Fluor 488-AffiniPure Goat Anti-Rabbit IgG (111-545-003). Hoechst 33342 was used for nucleus staining. Images were captured by Zeiss microscope with CCD.
For acrosome staining of tissue sections, after blocking in 5% BSA, slides were incubated with Alexa Fluor 488-lectin-PNA (20 μg/ml, Invitrogen, L21409) for 30 min, and then washed by PBS for three times. For acrosome staining of spermatozoa, slides were fixed by 4% PFA, permeabilized with 1% Triton X-100 for 5 min, incubated with 20 μg/ml Alexa Fluor 488-lectin-PNA at 37°C for 30 min, and then washed by PBS.

hESC-RPEs, for immunocytochemistry, were cultured on sterile, Matrigel-coated, #1.5 coverslips (GG121.5PRE; Neuvitro, Vancouver, WA, USA) prior to fixation with 4% methanol-free formaldehyde in PBS for 20 minutes. Cells were subsequently permeabilized with 0.1% Triton X-100 for 10 minutes, blocked with 5% goat serum (Jackson ImmunoResearch, West Grove, PA, USA) and 1% BSA (Thermo Fisher Scientific) in PBS for 30 minutes, and incubated in the following primary antibodies overnight at 4°C: rabbit–anti-ZO1 (40–2200; Thermo Fisher Scientific, 1:200) and mouse–anti-RPE65 (MAB5428; Millipore-Sigma, Burlington, MA, USA, 1:200). Coverslips were washed three times with PBS followed by secondary antibody incubation in blocking buffer for 1 hour at room temperature. Secondary antibodies were Alexa Fluor 594 AffiniPure goat anti-rabbit IgG (111585144; Jackson ImmunoResearch, 1:200) and Alexa Fluor 488 AffiniPure goat anti-mouse IgG (115545062; Jackson ImmunoResearch, 1:200). Nuclei were stained for 10 minutes with Hoechst 33342 in PBS (2 µg/mL), washed three times with PBS, mounted in ProLong Gold antifade, and imaged on an Olympus FV1000 Spectral Confocal with a PLAPON-SC 60X oil objective (NA: 1.40) and excitation laser lines at 405, 488, 559, and 635 nm. Scale bars for fluorescence confocal images are 50 µm.