Mowiol 4 88 dabco

Sections from two regions of the heart (heart base and apex) were examined. Sections of each subgroup (dose rate, age) were stained together with samples from sham-irradiated control animals and negative staining controls.
Specimens were stained with hematoxylin/eosin (HE) and sirius red following standard protocols. For specific immune fluorescence staining, sections were fixed in ice-cold ethanol/acetone, 50/50, for 10 min. Specimens were then washed with PBS (3 min) and incubated in blocking solution (PBS, bovine serum albumin 2%) for 20 min. Primary antibodies or isotype specific Ig (negative staining controls) were applied for 1 h diluted in blocking solution. Specimens were washed again, and corresponding, fluorescence-labeled, secondary antibodies were added (Table 2). After 45 min, specimens were rinsed again in PBS and incubated with DAPI nuclear stain for 5 min (Sigma Aldrich, Saint Louis, USA). Sections were embedded in Mowiol 4-88/DABCO (Carl Roth GmbH, Karlsruhe, Germany). Lectin-stained heart sections (fluroescein lycopersicon esculentum tomato lectin, FL-1171, Vector laboratories) were kindly provided by Fiona Stewart’s laboratory, NKI (The Netherlands Cancer Institute).

The GL261 cells were seeded in 8-well Permanox chamber slides (10,000 cells/well) and allowed to adhere. IFNγ (Recombinant Murine IFNγ, PeproTech) at a concentration of 50 ng/mL was added to the appropriate chambers after seeding. After 24 h, the chambers were removed and the cells were fixed with ethanol (96% v/v) for 15 min. The cells were permeabilized with 0.5% Triton-X 100 (Sigma-Aldrich) in PBS for 5 min and blocked with blocking solution (PBS, 0.25% Triton-X 100) containing 10% goat serum for 30 min at RT. Subsequently, the samples were incubated with the following primary antibodies overnight at 4 °C: PD-L1 (rabbit anti-mouse PD-L1, Abcam, Cambridge, UK) at a dilution of 1:50 (in PBS, 2% goat serum, 0.25% Triton-X 100) or Rabbit IgG Isotype Control Antibody (Abcam) at a solution of 1:170. The slides were washed three times with PBS and the cells were incubated with goat anti-rabbit AlexaFluor568 antibody (Invitrogen, ThermoFisher) at a solution of 1:1000 (in PBS, 2% goat serum, 0.25% Triton-X 100) for 1 h at RT followed by washing with PBS and nuclear staining with 4′, 6-diamidino-2-phenylindole (DAPI, 1:10,000 in distilled water). Slides were covered using Mowiol^®4-88/DABCO (CarlRoth, Karlsruhe, Germany). Fluorescence microscopy was performed using an Axiolab microscope (Zeiss) and ZEN lite 2.1, Zeiss, software.