Dylight 488 goat anti rabbit igg

Rats were sacrificed 3 or 7 days after suturing. The eyes were removed, and corneas were dissected from the ocular bulb. Corneas were embedded in SAKURA Tissue-Tek® O·C.T. compound and sliced at a thickness of 3 μm using a cryostat (Leica CM1950, China). After rewarming the frozen cross sections at room temperature, the sections were fixed in 4 % paraformaldehyde for 20 min, followed by a blocking and permeabilization step for 1 h in PBS with 1 % BSA and 0.5 % Triton-X. Then the sections were incubated with primary antibodies against ZO-1 (1:1000, 21773-1-AP, Proteintech, China), E-cadherin (1:100, 20874-1-AP, Proteintech, China) and β-catenin (1:200, 51067-2-AP, Proteintech, China) at 4 °C overnight. The sections were then incubated with Dylight 488 goat anti-rabbit-IgG (1:500, Abbkine, China) for 1h at room temperature. DAPI was used as a nuclear counterstain. The sections were photographed under a fluorescence microscope (Olympus, Japan).

Cells were seeded on to coverslips, irradiated with UVB light, and harvested at various time points. Afterward, 4% formaldehyde solution (15 minutes, room temperature) was act on fixed cells, the cell membranes were permeated with 1% Triton X-100 for 10 minutes, blocked with 5% goat or donkey serum for 60 minutes at 37°C, and cultivated overnight at 4°C with primary antibodies. The next morning, the cells were reacted at 37°C for 1 hour with the fluorescent secondary antibodies after washing 3 times. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology, China) for 5 minutes in the dark. All cell samples were imaged using laser confocal microscope (LSM 800; Carl Zeiss AG, Germany). The antibodies in this study: Hsp27 (1: 200; Cell Signaling Technology, USA) and p21 (1: 200; Abcam, UK), Alexa Fluor 594-conjugated goat anti-mouse IgG (1: 200; catalog no. SA00006-3; Proteintech, China), Dylight 488 goat anti-rabbit IgG (1: 200; catalog no. A23230-1; Abbkine Scientific, China), and Dylight 549 goat anti-rabbit IgG (1: 200; catalog no. A23320-1; Abbkine Scientific, China.).

Chlamydomonas or IMCD3 cells were fixed with methanol at −20 °C for 20 min or 4% paraformaldehyde/phosphate-buffered saline (PBS) for 15 min at room temperature, respectively. Cells were washed three times with PBS and permeabilized by incubation with 0.5% Triton X-100 in PBS for 15 min. IMCD3 cells were incubated on ice for 30 min to depolymerize cytoskeletal MTs before fixation. Nonspecific binding sites were blocked with 4% bovine serum albumin (BSA) for 1 h at 37 °C. Cells were incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used for immunofluorescence: antiacetylated-tubulin rabbit IgG (1:1,000, catalog [Cat.] #ab179484, Abcam) and anticeramide mouse IgG (1:100, Cat. #C8104-50TST, Sigma-Aldrich). Dylight 488 goat anti-rabbit IgG (1:500, Cat. #A23220-1, Abbkine), Dylight 549 goat anti-rabbit IgG (1:500, Cat. #A23320-1, Abbkine ), Dylight 488 goat anti-mouse IgG (1:500, Cat. #A23210-1, Abbkine), and Dylight 549 goat anti-mouse IgG (1:500, Cat. #A23310-1, Abbkine) were diluted 1:500 in 4% BSA/PBS and cells were incubated for 1 h at 37 °C. DAPI (Sigma-Aldrich) was used to visualize the nuclei.