Cx43 biological microscope

The method has been published previously (15 (link)). Briefly, the mammary tumor tissues were fixed with paraformaldehyde (PFA) and embedded in paraffin. Tissue blocks were cut into 5 μm thick sections in a HM 325 rotary microtome (Thermo Scientific). Sections were then deparaffinized and placed in the antigen retrieval solution (Histo-VT One, Nacalai USA). After rinsing thoroughly in 0.1 M PBS, the sections were pre-incubated in a blocking solution and then incubated with mouse PD-L1/B7- H1 antibody (R&D systems). After that, the sections were rinsed and incubated with anti-mouse biotinylated secondary antibody IgG (H+L) (Vector Laboratories). After washing with 0.1 M PBS, the sections were incubated with Avidin/Biotin complex (Vectastain ABC Elite kit; Vector Laboratories) and then chromagen DAB (3′,3′ -diaminobenzidine, Vector Laboratories). The sections were observed under an Olympus CX43 biological microscope. For quantitative assessment of PD-L1 staining, the average optical density (OD) of the regions of interest was measured by Fiji Image J software.

Tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned at a thickness of 5 μm. The sections for immunohistochemical staining were incubated with primary antibody to 4-Hydroxynonenal (1:200, Abcam, Cambridge, UK) overnight at 4ºC and secondary anti-goat IgG (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. A streptavidin-horse radish peroxidase (HRP) detection system was used to detect the antigen and sections were visualized with diaminobenzidine tetrahydrochloride. For immunofluorescence, sections were processed using rabbit anti-mouse LaminA primary antibody (1:200, Sigma-Aldrich, St. Louis, MO, USA) or rabbit anti-mouse xCT primary antibody (1:200, Abcam, Cambridge, UK), and Cy3- or Alexa Fluor 488-labeled anti-mouse IgG antibody (both from R&D Systems, Minneapolis, MN, USA) and DAPI (1 mg/ml, Roche, Basel, Switzerland). Stained sections were visualized using a fluorescence microscope (CX43 Biological Microscope, Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).