Cy3 labeled goat anti rabbit igg

The mice were decapitated and the heads were fixed with 4% paraformaldehyde for 2 days and decalcified for 21 days in 10% ethylene diamine tetra acetic acid (EDTA).Then the samples were paraffin-embedded and serially sectioned. The sections were stained with PAS for counting goblet cell numbers, stained with toluidine blue for assessing mast cell infiltration, and stained with immunofluorescence for assessing KCa3.1 immunolabelling. Following deparaffinization and rehydration, the sections were rinsed using PBS for three times and then microwaved for antigen retrieval in EDTA buffer (PH8.0). The sections were incubated with primary antibody (rabbit anti-mouse KCa3.1, 1:100 dilution, Abcam) at 37 °C for 60 min, rinsed for three times and incubated with secondary antibody (cy3-labeled goat anti-rabbit IgG, 1:300 dilution, Abcam) for 30 min. Then, 4,6-diamidino-2-phenylindole (DAPI) was added for nuclear staining. Goblet cell, mast cell and KCa3.1 positive cell numbers were microscopically counted under high-powered field (HPF, ×400), and five fields of each section were randomly selected and mean value per HPF was calculated. All pathology slides images were reviewed independently by three blinded reviewers whose inter-interviewer variability was not statistically significant.

The collected lung tissues were cut into small pieces and grinded to obtain cell suspension. Subsequently, CD45⁻ and CD31⁺ PMVECs selected by the CD45 and CD31 magnetic beads (Miltenyi Biotec, Gladbach, Germany; 10 μL beads/10⁷ cells) were inoculated onto Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Biosharp, Hefei, China) supplemented with 10% fetal bovine serum (FBS) in 5% CO₂ at 37℃. Immunofluorescence was used to detect the expression of CD31 and vascular endothelial cadherin (VE-cadherin) in the PMVECs. Briefly, the PMVECs were fixed in 4% paraformaldehyde (Sinoreagent, Shanghai, China) and permeabilized in 0.1% Triton X-100 (Beyotime Biotech, Shanghai, China). The PMVECs were incubated with anti-rabbit CD31 (1:100; Abclonal, Shanghai, China) and VE-cadherin (Abclonal, Shanghai, China) at 4℃ overnight. Then, the cells were incubated with Cy3-labeled goat anti-rabbit IgG (1:1000; Abcam, Cambridge, UK) at room temperature for one hour. 2-(4-amidinophenyl) 6-indolecarbamidine dihydrochloride (DAPI; Aladdin, Shanghai, China) was used for counterstaining nuclei. The images were captured using a fluorescence microscope (Olympus, Japan).

SMMC-7721 cells were cultured on coverslips. FITC–phalloidin was used to probe the samples for 1 h to analyze the actin remodeling. Additional coverslips were incubated overnight at 4°C with primary antibodies (diluted 1:50) directed against E-cadherin and vimentin. After the cells were washed, they were exposed to FITC-conjugated secondary antibodies (1:1000; Invitrogen, Carlsbad, CA, M30101, L42001), and then stained with DAPI for 20 min. The coverslips were washed and mounted. All tissue samples were embedded in paraffin blocks and then sectioned. Immunofluorescence staining was performed on 6 μm paraffin sections of the tumor tissue, which was blocked with 20% goat serum and then incubated with anti-mouse vegf mAb 1/1000 (Abcam, USA), anti-mouse ctgf 1/1000 (Abcam, USA) or anti-mouse ET-1 1/1000 (Abcam, USA) at 4°C overnight. To detect the bound antibodies, CY3-labeled goat anti-rabbit IgG at 1/1000 (Abcam, USA) or FITC-labeled goat anti-mouse at 1/200 (Abcam, USA) was used at 22°C for 45 min. Nuclear staining was performed with DAPI at a 1/1000 dilution (Abcam, USA) at 22°C for 10 min. At least three sections per mouse were examined for each immunostaining condition. Images were acquired using a confocal microscope.

The segments of spinal cord were harvested, post-fixed in 4% paraformaldehyde for 4 h, and then stored overnight at 4 °C in 0.2 M phosphate buffer. The samples were subsequently cryoprotected in 30% sucrose for 48 h. Segments were embedded in OCT (VWR International), and frozen sectioned at 10 μm. After being blocked with 0.01 M PBS containing 3% BSA, 0.1% Triton X-100, and 10% normal goat serum for 1 h at 37 °C, the sections were incubated overnight at 4 °C with primary antibodies: GFAP (1:400, Sigma); PAR1 (1:400, Novusbio). Thereafter, the sections were washed with PBS and incubated with the Cy3-labeled goat anti-rabbit IgG (1:400, Abcam) or the Alexa Fluor 488-labeled donkey anti-mouse IgG (1:400, Abcam). Sections were photographed with fluorescence microscope (ZAISS, axio image M2).