Alexa fluor 594 conjugated igg secondary antibody

A total of 1.5 × 10⁶ SH-SY5Y cells were seeded in the camber slide and incubated in a 5% CO₂ incubator at 37 °C for 24 h. We treated VPA (0, 1, 5, and 10 mM), 100 nM bafilomycin A1, or 5 mM VPA plus 100 nM bafilomycin A1 to the SH-SY5Y cells for 24 h. The cells were fixed with PBS containing 2% formaldehyde and 2.5% glutaraldehyde for 10 min, washed three times with 1× PBS. Blocking was performed using a blocking solution (PBS with 0.1% Tween 20 and 1% bovine serum albumin) at room temperature for 1 h. The cells treated with VPA (0, 1, 5 and 10 mM) were incubated with anti-FOXO3a primary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The cells treated with VPA (0, 5 mM), 100 nM bafilomycin A1, or 5 mM VPA plus 100 nM bafilomycin A1 were incubated with anti-LC3A/B primary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. After washing three times with 1 × PBS, the cells were incubated with Alexa Fluor 594-conjugated IgG secondary antibody (Abcam, Cambridge, UK) in the dark for 1 h at room temperature. After washing three times with 1 × PBS, the cells were mounted using Fluoroshield mounting solution (Abcam, Cambridge, UK). A fluorescence microscope (Korea Lab Tech, Seongnam, Gyeonggi, Korea) was used for image confirmation.

Immunofluorescence staining were performed on paraffin sections of colon tissue. After dewaxing and rehydration, antigen retrieval was performed in sodium citrate buffer (pH 6.0) for 3 min by microwaving the sections. Sections were then blocked using 1% sheep serum in PBS for 1 h at room temperature and immunostained with primary antibodies overnight at 4°C. The primary antibodies from Cell Signaling Technology including Claudin-1 and ZO-1. Afterward, sections were washed by PBST and incubated with Alexa Fluor 594-conjugated IgG secondary antibody (1:1000 dilution, Abcam, MA, United States, cat#ab150116) for 2 h at room temperature, followed by counterstaining with DAPI (10 μg/ml) for 5 min, and mounted using antifade solution. Finally, sections were photographed under a fluorescence microscope (Olympus, Tokyo, Japan).