Anti uchl3 rabbit polyclonal antibody

UCHL3 protein was analyzed by western blot [18 (link)]. 1.0 × 10⁷ spermatozoa were lysed in 200 μL cold lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65mM DTT, 1mM PMSF, and 1 × proteinase cocktail). The extracts (50 μg protein) were loaded into each lane. The primary antibodies used included anti-UCHL3 rabbit polyclonal antibody (1:500; Bioss, Beijing, China), and anti-α-tubulin rabbit monoclonal antibody (1:5,000; ab52866, Abcam Inc., Cambridge, MA, USA). The secondary antibody used was the HRP-labelled goat anti-rabbit IgG (1:5,000; 111-035-003; Jackson ImmunoResearch Laboratories Inc., WestGrove, PA, USA). The band density was quantified using the ImageJ software (rsbweb.nih.gov/ij/).

Smeared semen was fixed in 100% acetone for 15 min at 4°C and stored at –80°C until use. After thawing and warming up to room temperature, the smears were permeabilised with 1% Triton X-100 in phosphate buffered saline (PBS), pH 7.4, for 15 min. After washing three times for 15min with washing buffer [0.2% Tween 20 and 1% inactivated normal goat serum (INGS) in PBS], 5% INGS in PBS was added to block nonspecific sites on the spermatozoa for 50min. Anti-UCHL3 rabbit polyclonal antibody (1:50, Bioss, Beijing, China) was added, and the slides were incubated at 4°C overnight. The antibody diluent was PBS containing 0.5% Tween 20 and 1% INGS. Slides were washed with washing buffer more than three times for 15 min, and then the Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (1:2,000; Molecular Probes, Invitrogen, Carlsbad, CA, USA) was added. Thereafter, slides were washed once with washing buffer (10 min) and twice with PBS (10 min). The nucleus was stained with anti-fade VECTASHIELD^® Mounting Medium, including DAPI (H-1200; Vector Laboratories Inc, Burlingame, CA, USA).
The negative control was performed in the same way by substituting non-immune rabbit serum for the primary antibody. Slides were observed under a confocal laser scanning microscope (Leica TCS-SP5, Leica Microsystems, Wetzlar, Germany).