Hpa021935

The antibodies used in western blotting, Co-IP and immunofluorescence staining are as follows: anti-QRICH2 (1:200, sc-514279, Santa Cruz Biotechnology), anti-QRICH2 (1:50, HPA052219, Sigma-Aldrich) for immunofluorescence staining of mice sample, anti-QRICH2 (1:50, HPA021935, Sigma-Aldrich) for immunofluorescence staining of human sample, anti-AKAP3 (1:100, provided by Prof. Huayu Qi) for immunofluorescence staining, anti-AKAP3 (1:100, 13907-1-AP, Proteintech) for Co-IP, anti-ODF2 (1:500, 12058-1-AP, Proteintech), anti-CABYR (1:500, 12351-1-AP, Proteintech), anti-ROPN1 (1:200, sc-130455, Santa Cruz Biotechnology), MARCH10 (1:500, bs-10732R, Bioss), anti-TSSK4 (1:400, A7861, ABclonal), anti-ubiquitin (1:1000, ab7780, Abcam), anti-Flag Tag (1:500, 66008-2-Ig, Proteintech), anti-Myc Tag (1:1000, sc-764, Santa Cruz Biotechnology), anti-α-Tubulin (1:2000, T7451, Sigma-Aldrich) and anti-GAPDH (1:5000, ab8245, Abcam). Anti-AKAP3 mouse monoclonal antibody for western blotting was kindly provided by Prof. Huayu Qi. Anti-TSSK4 rabbit polyclonal antibody for Co-IP was kindly provided by Prof. Long Yu.

Sperm cells were fixed and immunostained with rabbit anti-QRICH2 (1:50, HPA021935, Sigma-Aldrich) and mouse anti-α-Tubulin (1:100, T7451, Sigma-Aldrich) overnight at 4 °C. After washed in 1× PBS, sperm cells were incubated with Atto 488 (1:500, 41051, Sigma-Aldrich) or Alexa fluor 568 (1:500, A10037, Thermo Fisher) secondary antibodies for 1 h at room temperature. Finally, the sperm samples were stained with imaging buffer (PBS with the addition of 100 mM mercaptoethylamine at pH 8.5, 5% glucose (w/v), 0.5 mg/ml glucose oxidase (Sigma-Aldrich), and 40 μg/ml catalase (Roche Applied Science)) and illuminated at 657 nm for imaging. The 501 nm and 568 nm lasers were used to activated Atto 488, and Alexa fluor 568, respectively. A cylindrical lens (focal length = 1 m) used for 3D localization was inserted into the detection path to enable determination of z positions from the ellipticities of the molecular images and the x and y positions from the centroid positions.