Fitc goat anti rabbit igg h l

Sperm immunocytochemistry was carried out as previously explained [64 (link)]. Briefly, sperm were fixed in 4% paraformaldehyde at room temperature (RT) for 20 min and then centrifuged at 1200× g for 6 min. The pellet was resuspended in 1x PBS (pH = 7.3). Sperm smears were made on poly-D-lysine-coated coverslips and allowed to dry at RT. Dry smears were washed out three times for 5 min each with PBS. Smears were then blocked with 5% BSA in PBS at 4 °C for 2 h. Smears were washed out again with PBS 3 times (5 min for each washout). Primary antibodies, KCNMA1 Rabbit pAb (Cat # A15283, ABclonal, Woburn, MA 01801, USA) and KCNU1 Rabbit pAb (Cat # A14967, ABclonal), were diluted to 1:100 in 5% BSA-PBS, and the washed smears were incubated with the diluted primary antibodies at 4 °C for overnight. After overnight incubation, smears were washed out with PBS 3 times and incubated with the secondary antibody, FITC Goat Anti-Rabbit IgG (H+L) (Cat# AS011, ABclonal), diluted to 1:200 in 5% BSA-PBS for 75 min at RT under dark conditions. Smears were thoroughly washed using PBS and then air-dried at RT before mounting with the permanent mounting reagent. Photographs of the immunolabeled sperm were obtained using confocal microscopy (Leica TCS SP8, Leica Biosystems Nussloch GmbH, Nußloch, Germany).

Paraffin sections of mouse AD model skin were prepared as previously described. Stained with hematoxylin and eosin (H&E) to determine the thickening and cellular infiltration of the skin. Furthermore, the immunofluorescence procedure was performed in the same way as shown in macrophage localization. F4/80 Rabbit pAb (1:200) (ABclonal, Wuhan, China) was employed as the primary antibody and FITC Goat Anti-Rabbit IgG (H+L) (1:400) (ABclonal, Wuhan, China) as the secondary antibody to observe macrophage infiltration in experimental mice. A digital section microscope was used to monitor and photograph both H&E and F4/80 practical sections.

The cells were plated into 12-well plates and fixed with 3% formaldehyde (Sinopharm, China) for 15 min. After washing three times with PBS, the cells were permeabilized with Triton-X 100 (1%) for 10 min. Subsequently, cells were blocked with bovine serum albumin (3%) for 30 min. Next, cells were labeled with primary antibodies including anti-caspase-1 (1:500; AF5418, Affinity, USA), anti-ASC (1:100; DF6304, Affinity), anti-DDX3X (1:50; ab271002, Abcam, UK), anti-SQSTM1 (1 μg/mL; ab109012, Abcam), anti-HMGB1 (1 μg/mL; ab18256, Abcam), and anti-NLRP3 (1:100; ab263899, Abcam) overnight at 4° C. Following that, cells were incubated with the secondary antibody FITC goat anti-rabbit IgG (H+L) (ABclonal, MA, USA) and 4’,6-diamidino-2-phenylindole (DAPI, 1:500) for 1 h in the dark. Finally, the cells were visualized using an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer, MA, USA).

BmPiezo antibodies (BmPiezo-R) were generated in rabbits using peptide-containing the amino acid residues (RPKEEPEEQRALPPSRSERS) at the end of the C-terminal of BmPiezo and affinity-purified at ABclonal. Immunofluorescence staining experiments were performed using sperm bundles isolated from excised testes. The collected sperm were fixed in permeabilizing buffer (1 × PBS + 4% paraformaldehyde + 0.1% Triton X-100) for 15 min, washed in PBST three times, and subsequently incubated in blocking solution (1 × PBS + 0.1% Triton X-100 + 1% bovine serum albumin) for 60 min. A primary antibody was added to the blocking solution, and then the samples were incubated at 4 °C for 36 h. After five washes in PBST, samples were incubated with the secondary antibody, TRITC Phalloidin, and Hoechst for 2 h at room temperature; washed five times with PBST; and subsequently mounted in PBS. All images were taken on a Nikon FV1000 microscope. Antibodies and dilutions used were as follows: BmPiezo-R (ABclonal), 1:100; FITC goat anti-rabbit IgG (H+L) (ABclonal, Cat. AS011), 1:100; and TRITC Phalloidin (YEASEN), 1:200.

Fixed samples were incubated with PBS containing 0.5% Triton X-100 for 20 min. PBS was added and washed three times, followed by blocking with 5% BSA for 30 min. Sections were then washed, and primary antibodies (AIM2, A3356, 1:100, Abclonal, CN; Caspase-1, 22915-1-AP, 1:100, Proteintch, CN; NLRC4, A7382, 1: 100, Abclonal, CN) were added and then incubated overnight at 4°C. A secondary antibody (Cy3 GoatAntiRabbit IgG (H+L) (AS007, 1:200, Abclonal, CN) or FITC Goat AntiRabbit IgG (H+L) (AS011, 1:200, Abclonal, CN)) was added and incubated for 50 min at room temperature (23°C ± 2°C), and cell nuclei were stained with DAPI.