Sodium azide

For surface fluorescent immunostaining cells were incubated with Abs for 30 min on ice in PBA staining buffer (PBS containing 0.5% BSA (Serva, Heidelberg, Germany) and 0.01% sodium azide (AMRESCO, Inc. (VWR International, LLC), Aurora, CO, USA)) and then washed twice in the same buffer. Some samples were stained with Hoechst 33342 (FluoroPure^™ Grade, Thermo Fisher Scientific, USA) during 45 min on water bath (+37°C). Samples were analyzed in FACSCalibur flow cytometer (BD Biosciences, USA), equipped with 488 and 640 nm lasers. Samples labeled with Hoechst 33342 were analyzed on FACSVantage DiVa machine (BD Biosciences, San Jose, CA, USA) equipped with 405, 488, and 643 nm lasers. From 50,000 to 300,000 events for freshly isolated NK cells and from 5000 to 30,000 events for NK cell fractions and clones in FSC-SSC gate were recorded.

All tissues were dissected 4h after the last post-drug treatment, passing the KXA half-life of 2–3 h (Zhang et al., 2018 ). For histological analysis, animals were quickly anesthetized with isoflurane (Zoetis, Cat#6089373) and secured to the perfusion plate. The chest was open to expose the heart. The left ventricle was cannulated and the inferior vena cava cut. The animals were initially perfused with 20 ml of phosphate-buffered saline (PBS) with heparin (100 mg/l, Sigma, Cat#H0878), followed by 20 ml of 4% (w/v) paraformaldehyde (PFA, Sigma, Cat#P6148) in PBS using a peristaltic pump (Behr, Cat#PLP 380, speed: 25 rpm). The animals were decapitated, the retina and brain explanted and post-fixed in 4% (w/v) PFA/PBS for 30 minutes and overnight (16h), respectively. Then the tissues were washed in PBS and stored at 4°C with 0.025% (w/v) sodium azide (VWR, Cat#786–299). For cryoprotection, the tissue was transferred to 30% (w/v) sucrose (Sigma, Cat#84097) in PBS and incubated overnight at 4°C. To increase antibody permeability, the brain slices were frozen over dry-ice and thawed at room temperature for three cycles. Then, the brain was sliced in 100 μm coronal slices on a vibratome (Leica VT 1200S), if not otherwise indicated. For parasagittal sections, the brain was divided along the midline and the same vibratome settings were used.