Alexa fluor 594 goat anti rabbit

Fresh colorectal cancer tissues were cryosectioned and fixed in 4% paraformaldehyde and then immunostained following the procedures of a previous study (21 (link)) with a primary antibody against tricellulin (product no. 48-8400; Invitrogen; Thermo Fisher Scientific, Inc.) at a dilution of 1:200 at 4°C overnight. Subsequently, the sections were washed and incubated with the respective secondary antibody (Alexa Fluor^® 594 goat anti-rabbit; product no. 8889S; 1:1,000; Cell Signaling Technology, Inc.) and 4′,6-diamidino-2-phenylindole (DAPI) (Wuhan Boster Biological Technology, Ltd.).
To visualize the cell actin cytoskeleton, FITC-Phalloidin was utilized to stain HCT116 cells (NC, TRIC-KD, and TRIC-OE). In brief, cells (1×10⁴) were seeded onto coverslips and grown overnight and then fixed and permeabilized in the same way as the cryosections. The cells were next incubated with the Alexa Fluor^® 488 Phalloidin working solution (Beijing Solarbio Science & Technology, Inc.) for 30 min at room temperature in the dark and then with DAPI for 10 min. Images were obtained from the stained cells with a fluorescence Olympus BX53F microscope (magnification, ×600; Olympus Corporation).

Tissue sections were incubated in 0.3% Triton X-100 for 20 min and then blocked with 10% goat serum for 45 min at room temperature (20° to 23°C). The sections were incubated with primary antibodies, including anti–c-Fos (1:1000; Abcam, ab190289), anti-MOR (1:300; EMD Millipore, AB5509), anti-CCK2R (1:500; Alomone Labs, ACR-042), anti-CCK8s (1:300; IMMUNOSTAR, 20078), anti-NeuN (1:1000; Sigma-Aldrich, MAB377), and anti-CaMKII (1:250; Abcam, ab22609) at 4°C for 24 hours. After the incubation with primary antibodies, sections were washed with PBS (5 × 8 min) and then incubated with corresponding fluorophore-conjugated secondary antibodies, including Alexa Fluor 594 goat anti-rabbit (1:500; Cell Signaling Technology, 8889S), Alexa Fluor 594 goat anti-pig (1:300; Invitrogen, A11075), Alexa Fluor 488 goat anti-mouse (1:500; Cell Signaling Technology, 4880S), Alexa Fluor 488 goat anti-rabbit (1:500; Cell Signaling Technology, 4412S), and Alexa Fluor 594 goat anti-mouse (1:500; Cell Signaling Technology, 8890S) for 2 hours at room temperature. For c-Fos immunostaining, HC-mice and CC-mice were anesthetized in the HC and CC 90 min after the last morphine injection on day 5, respectively, followed by transcardially perfusion. Brian slices were examined and photographed using an LSM 880 (Zeiss, Germany) confocal microscope.