Fluorescein isothiocyanate fitc conjugated goat anti rabbit igg antibody

Caco-2 cells (5 × 10⁵ cells/well) were cultured and treated for 48 h with the control medium, medium with LPS (1 μg/mL), and medium with LPS (1 μg/mL) and BWI mix (10⁷ CFU/mL). After being washed with PBS, Caco-2 cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min and permeabilized with PBS containing 0.25% Triton X-100 for 10 min at 25 °C. Next, Caco-2 cells were incubated with 1% BSA in PBS for 30 min at room temperature. The primary antibody occludin (Cell Signaling, Danvers, MA, USA, cat. no. #5506) was diluted 1:20 and incubated for 1 h overnight at 4 °C and a secondary antibody (fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich, St. Louis, MO, USA, cat. no. F9887) was diluted to 1:100 and incubated for 30 min at room temperature. Finally, 300 nM DAPI solution (Invitrogen, Waltham, MA, USA, cat. no. D1306) was added to Caco-2 cells and incubated for 10 min at room temperature for nuclear counterstaining. After mounting with a mounting solution (Invitrogen, Waltham, MA, USA, cat. no. 00-4958-02), images were acquired using an Observer D1 inverted microscope (Carl Zeiss, Jena, Thuringia, Germany) equipped with Automatic Component Recognition module and AxioVision 3.1 software (Carl Zeiss, Jena, Thuringia, Germany) using a 40×/1.0 objective lens.

Rabbit-anti-Wilms’ tumor 1 (WT1) antibody was obtained from Abcam (Hong Kong, China) and mouse-anti-synaptopodin antibody was obtained from Acris Biotechnology (Herford, Germany). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody were obtained from Sigma Chemical Co. (St. Louis, MO, USA). FITC-conjugated rabbit anti-mouse IgG antibody and rabbit anti-mouse complement C3 antibody were obtained from Beijing Biosynthesis Biotechnology Co. LTD. (Beijing, China).