Fitc labeled goat anti rabbit secondary antibody

The colon tissue sections were washed 3 times with PBS. Citric acid (pH = 6.0) was used for antigen repair for 5 min. Sections were blocked with 10% normal goat serum for 30 min and incubated with the antibodies against TRPV1 (1 : 100, Abcam, UK) and NF-κB p65 (1 : 200, Proteintech, China) at 4°C overnight. Subsequently, the sections were incubated with the CY3-labeled goat anti-mouse secondary antibody (1 : 800, Proteintech, China) and FITC-labeled goat anti-rabbit secondary antibody (1 : 400, Proteintech, China) for 1 h, and nucleus was stained with DAPI.

The sections were fixed and permeabilized with 0.3% Triton (Solarbio Life Sciences) for 10 min. After blocking non-specific binding using 5% BSA, sections were incubated with LC3 (1:100, Cat No. 14600-1-AP, Proteintech Group, Inc), E-cadherin (1:100; Cat No. 20874-1-AP, Proteintech Group, Inc.), and vimentin (1:100; Catalog number: 10366-1-AP, Proteintech Group, Inc.) antibodies overnight at 4 °C. After washing with PBS three times, sections were incubated with FITC-labeled goat anti-rabbit secondary antibody (1:50; Catalog number: SA00003-2, Proteintech Group, Inc.). The cell nuclei were stained with DAPI, and the expression of target proteins was visualized using a fluorescence microscope. For confocal microscopy, after transfecting RFP-GFP-LC3 adenovirus for 48 h, PC cells were fixed, and autophagosome dots were photographed and counted using a laser scanning confocal microscope.