Alexa fluor 488 fluorescent secondary antibody

Recombinant rat CTLA‐4 protein was purchased from Sino Biological (Wayne, PA, USA). After reconstitution, the recombinant protein was conjugated to flow cytometry beads using the Functional Bead Conjugation kit from Becton Dickinson (Wokingham, UK). Samples were stained with the 9H10 anti‐CTLA‐4 antibody and the relevant isotype control then with an Alexa Fluor 488 fluorescent secondary antibody (Thermo Fisher Scientific, Hemel Hempstead, UK) and assessed using flow cytometry. Recombinant rat PD‐1 protein was purchased from Sino Biological (Wyne, PA, USA), and cross‐reactivity has been previously validated (Smith et al, 2019).

Samples (n = 5–6) were fixed with 10 (v/v)% formalin and permeabilized with 0.1 (v/v)% X-100 Triton (ThermoFisher Scientific, Waltham, MA, USA)/PBS for 10 min at RT. After PBS rinsing, non-specific binding was blocked with 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at RT. Then, samples were incubated overnight at 4 °C with primary antibodies diluted in 0.1% BSA/PBS against ACTA2 (rabbit, ab32575, Abcam, Cambridge, UK), and anti-TNMD (rabbit polyclonal) generated against TNMD C-terminus (237-317 aa) provided by Prof. Denitsa Docheva (produced in co-operation with Metabion International, Planegg, Germany, PAB 201603-00002). Subsequently, samples were washed in PBS, incubated with 0.3% (v/v) hydrogen peroxide (PanReac AppliChem, Barcelona, Spain) for 15 min at RT, and incubated with anti-rabbit AlexaFluor 488 fluorescent secondary antibody (ThermoFisher Scientific, Waltham, MA, USA) for 1 h at RT. Finally, nuclei and F-actin filaments were counterstained with DAPI and phalloidin for 30 min at RT, respectively. Immunolabeled samples were analyzed through confocal laser scanning microscopy CLSM, TCS SP8, Leica, Wetzlar, Germany) and the quantitative analysis of the immunofluorescence images was performed using ImageJ 1.520 (NIH, Bethesda, MD, USA) software.