Cy3 labeled goat anti rabbit igg

To investigate co-localization of rTsSPc and receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells, the IIFT was performed as reported before [47 (link),48 (link)]. Briefly, confluent Caco-2 monolayers were apically incubated with 20 μg/ml of rTsSPc for 2 h at 37 °C. IIL ES antigens were used as a positive control and PBS as a negative control. After interaction, the cells were washed three times with washing buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 6.8) to eliminate unbound protein molecules. The cells were fixed in 4% paraformaldehyde at room temperature for 15 min. To reduce non specificity, cells were blocked for 2 h with 1% bovine serum albumin (BSA; Sigma). Then, the cells were probed overnight at 4 °C by rabbit anti-human RACK1 antibody (1:1 000; Servicebio, Wuhan, China) and mouse anti-rTsSPc immune serum (1:100). Then, the cells were stained by using Alexa Fluor 488 labeled goat anti-mouse IgG (1:100; Abways, Shanghai, China) and CY3 labeled goat anti-rabbit IgG (1:100; Servicebio) as the secondary antibody. After being washed with PBST, a 4′,6-diamidino-2-phenylindole (DAPI) was used for fluorescence staining of the cell nucleus. Finally, the cells were observed under a laser confocal fluorescence microscopy [24 (link),42 (link),49 (link)].

The following antibodies were used: anti-Bcl-2 pAb (Bioss, Proteintech Group), anti-Cytochrome C Rabbit pAb (ABclonal Technology), anti-Bax Rabbit pAb (Wanlei Biotechnology Co., Ltd), anti-Bak Rabbit pAb (ABclonal Technology), anti-caspase-3 Rabbit pAb (ABclonal Technology), anti-caspase7/cleaved-caspase7 Rabbit pAb (ABclonal Technology), IL-1β (Wanleibio Biotechnology Co., Ltd), anti-aggrecan Rabbit pAb (ABclonal Technology), Coll1A2 Rabbit pAb (ABclonal Technology), anti-GAPDH Rabbit pAb (ABclonal Technology), HRP goat anti-rabbit IgG (ABclonal Technology), Alexa Fluor® 488 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd), Cy3 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd).

The ERR granulation tissues were frozen and sliced, and then dried at room temperature. After washed with PBS, these cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% bovine serum albumin (BSA), and then incubated with primary antibodies overnight at 4 °C. Primary antibodies included αENaC (Thermo Fisher Scientific), and Fibronectin (Thermo Fisher Scientific). The secondary antibodies were respectively Cy3-labeled Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) and Alexa Flour-labeled Goat Anti-Mouse IgG (Servicebio). Nuclei were counterstained with DAPI (Cell Signaling Technology). Finally, the slides were imaged using a fluorescence microscope (Echo Revolve, San Diego, CA, USA) or a laser scanning confocal microscope (Leica-SP8, Wetzlar, Germany), and the colocalization of αENaC with Fibronectin was observed.