Texas red conjugated anti rabbit

Cells cultured in six-well plates were irradiated with a single dose of 8 Gy of ⁶⁰Co γ-rays. After 1 h or 48 h, cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min. Then, we washed cells with PBS and permeabilized the plasma membrane of cells with 0.1% Triton X-100 for 30 min at room temperature. Cells were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with E-cadherin, Vimentin (Cell Signaling Technology), α-SMA, TBK1, or p-AKT (Abcam) antibodies at 4 °C overnight. After being washed with PBS, the sections were incubated with Alexa Fluor 488-conjugated anti-mouse (Invitrogen) and Texas red-conjugated anti-rabbit (Vector Laboratories, Burlingame, CA, USA) antibodies at room temperature for 30 min. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and the slides were analyzed using a fluorescence microscope (Nikon, Tokyo, Japan). For the lung tissue, after deparaffinization and antigen retrieval, antibodies against E-cadherin and α-SMA were mixed and used for immunofluorescence staining.

The following antibodies were used in the current study: mouse monoclonal antibodies: CD31 (Abcam), Mac-2 (Santacruz, Germany), α-SMA (Boster, Wuhan, China); rabbit polyclonal antibodies: FKBP11 (Bioss, Beijing, China), p65, p-p65 (Cusabio, Wuhan, China), and MMP-9 (Santacruz, Heidelberg, Germany); VCAM-1, ICAM-1, MCP-1 (Wanleibio, Shenyang, China); β-actin (New England Biolabs, Germany); IL1-β (Sigma-Aldrich, Taufkirchen, Germany). The following HRP conjugated secondary antibodies were used for immunoblotting: rabbit IgG (New England Biolabs, Germany), mouse IgG (Abcam). The following secondary antibodies for immunofluorescence were used: Texas red conjugated anti-rabbit and FITC conjugated anti-mouse (Vector Laboratories, CA, USA).
The following reagents used in the current study were: Ang II (Sigma-Aldrich, St. Louis, MO), Trypsin-EDTA and HEPES (Gibco, Germany), fetal bovine serum (Sigma-Aldrich, St. Louis, MO), Protein A-Agarose Immunoprecipitation Reagent (Santacruz, Germany); phosphatase inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and protease inhibitor cocktail (Roche diagnostics GmbH, Mannheim, Germany); BCA reagent (Perbio Science, Bonn, Germany); Vectashield mounting medium with DAPI (Vector Laboratories, CA, USA); PVDF membrane and immobilion enhanced chemiluminescence reagent (Millipore GmbH, Germany).

The day before irradiation, cells were placed in 24-well plates at density 4 × 10⁵ cancer cells per well (monocultures) or 2 × 10⁵ cancer cells plus 2 × 10⁵ normal cells per well (co-cultures). Three days after, irradiation cells were detached by 0.25% trypsin, and cell apoptosis was detected using PE Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. In brief, cells were resuspended in binding buffer and stained with Annexin V and 7-AAD at room temperature for 15 min in dark. Finally, cells were analyzed using FACSCanto™ II flow cytometer. Additionally, one day later, cells were stained using anti-Cleaved Caspase-3 antibody (Cell Signaling Technology, Denvers, MA, USA) followed by secondary antibody Texas red-conjugated anti-rabbit (Vector Laboratories, Burlingame, CA, USA) and DAPI dye (Merck) administration. Microscopic observations were performed using an LSM710 confocal microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). The results were defined as the area occupied by stained cells calculated using ImageJ 1.48v software (National Institutes of Health, Bethesda, MD, USA).