Alexa fluor 568 labeled goat anti mouse igg

First, the control and Tubb4b-KO spermatozoa cells were fixed and ruptured membranes with DPBS, including 4% paraformaldehyde (P0099, Beyotime, Shanghai, China) 0.1% Triton X-100 (X100, Sigma, Shanghai, China) for 30 min. After washing 3 times with general DPBS, the cells were blocked in 1% BSA (A8806, Sigma, Shanghai, China) for 30 min. Then, cells were incubated with a mouse monoclonal anti-β-Tubulin antibody (T8328, Sigma, 1:1000) at 4 °C overnight. The next day, using general DPBS to wash 3 times again, the secondary antibody, Alexa Fluor 568-labeled goat anti- mouse IgG (A11031, Invitrogen, 1:100) was incubated at 37 °C in the dark for 1 h. Finally, continuing the general DPBS washing, cells were added 10 μg/mL Hoechst 33342 (62249, Thermo Scientific, Waltham, MA, USA) to stain the nuclei and observed the fluorescence under the fluorescence microscope.

The previously published gut immunocytochemistry protocol was used [16 (link)] with minor modifications. Briefly, whole abdomens were immunostained before the intestines were dissected out and mounted for imaging. Whole abdomens were separated and punctured for reagent infiltration to the intestine in phosphate buffered saline with 0.2% Triton X-100 (PBS-T, pH 7.2), and fixed in 4% paraformaldehyde in PBS-T overnight at 4°C. After 3 washes with PBS-T, samples were blocked with 3% goat serum in PBS-T for at least 30 min. Abdomens were incubated with primary antibodies or antisera for 1–2 days at 4°C, washed with PBS-T, and incubated with secondary antibodies overnight at 4°C. The primary antibodies used were rat anti-TRPA1 (1:200) [19 (link),22 (link),37 (link)], rabbit anti-GFP (1:1000, Life Technologies, CA, USA), anti-Prospero (1:10, Developmental Studies Hybridoma Bank at the University of Iowa). The secondary antibodies used were Alexa Fluor Cy3-labeled goat anti-rat (1:1000, Jackson Laboratory, ME, USA), Alexa Fluor 488-labeled goat anti-rabbit (1:200, Life Technologies, CA, USA), and Alexa Fluor 568-labeled goat anti-mouse IgG (1:1000, Life Technologies, CA, USA). A Zeiss LSM 700 laser-scanning confocal microscope was used to acquire images of immunostained samples.