Alexa fluor 488 conjugated goat anti mouse igg

A non‐tumourigenic immortalized adult human colon epithelial cell line, FHC, was obtained from the ATCC. The colon cancer cell lines SW620, LoVo, SW1116, SW480, HT‐29, DLD1, and LS174T cells were grown in RPMI 1640 containing 10% fetal bovine serum (GIBCO), 1% glutamine (Life Technologies), penicillin G (100 µ/mL) and streptomycin (100 μg/mL) (Sigma‐Aldrich). All the cell cultures were maintained as a monolayer culture at 37°C in a humidified atmosphere containing 5% CO₂.
Recombinant human TGF‐1 (240‐B) and monoclonal anti‐TGF‐β1 antibody was purchased from R&D Systems. Rabbit antibodies against HOXD9 (SAB4200029‐200UL), E‐cadherin (20874‐1‐AP), AKT (9272S) and ErK1/2 (#4695S), or mouse antibodies against CDK4 (#2906S) and CDK6 (#3136S) were purchased from Cell Signaling. Mouse antibody against vimentin (60330‐1‐lg) was purchased from proteintech. Mice antibodies against Cyclin B1 (sc‐245), Cyclin D1 (sc‐8396), MMP‐9 (sc‐21733) were purchased from Santa Cruz Biotechnology. Mouse antibody against β‐tubulin was purchased from Ray Antibody Biotech. Alexa Fluor ®488—Conjugated Goat anti‐ Rabbit IgG (conjugated to red fluorescent dyes, ZF‐0511)and Alexa Fluor ®488—Conjugated Goat anti‐Mouse IgG (conjugated to green fluorescent dyes, ZF‐0512) were purchased from ZSGB‐BIO, China.

Immunostaining of the spermatozoa was performed as described previously (Sha, Xu, et al., 2017). Briefly, the prepared spermatozoa were smeared onto poly‐L‐lysine coated slides, allowed to air‐dry, washed in phosphate‐buffered saline (PBS), fixed in 4% PFA (F8775, Sigma, United States) for 10 min at RT, and washed twice in PBS, followed by permeabilization with 0.2% Triton X‐100 (93443, Sigma, USA). Then, the samples were blocked with PBS containing 1% bovine serum albumin (A1933, Sigma, USA) and 2% normal goat serum (NS02L, Millipore, USA) for 30 min at RT. Slides were incubated with rabbit anti‐EIF4G1 (15704‐1‐AP, Proteintech, USA), rabbit anti‐COXIV (11242‐1‐AP, Proteintech, USA), or rabbit anti‐ATP6 (55313‐1‐AP, Proteintech, USA) primary antibodies and costained with anti‐acetylated tubulin (66200‐1‐Ig, Proteintech, USA) primary antibodies 1 hr at RT, followed by incubation with Alexa Fluor^® 594‐conjugated goat anti‐rabbit IgG (ZF‐0516, zsbio, China) and Alexa Fluor^® 488‐conjugated goat anti‐mouse IgG (ZF‐0512, zsbio, China) secondary antibodies for 45 min at RT. Slides were subsequently washed three times in PBS, mounted with Vectashield containing DAPI (Vector Laboratories) and examined under a laser scanning confocal immunofluorescence microscope (LSM510 Exiter, Carl Zeiss, Germany). The specific antibodies used in this assay are listed in Table S2.

Prepared sections were blocked with 3% BSA for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. After washing with PBST, the samples were incubated with the secondary antibodies at a 1:200 dilution for 1 h at 37°C. The slides were subsequently mounted with Vectashield containing DAPI (H-1200, Vector Laboratories, United States). The primary antibodies included Scp1 (ab15090, Abcam, United States), Scp3 (ab97672, Abcam, United States), cleaved caspase-3 (#9661, CST, USA), and Ptbp2 (ab154787, Abcam, United States). The secondary antibodies were Alexa Fluor® 594-conjugated goat anti-rabbit IgG (ZF-0516, ZSGB-BIO, China) and Alexa Fluor® 488-conjugated goat anti-mouse IgG (ZF-0512, ZSGB-BIO, China).