Zen 2.3 blue imaging software

In the microfluidic migration experiment, NK cells were immobilized with 4% paraformaldehyde at 10 min after the addition of chemoattractant solutions. Thirty minutes later, the NK cells were washed once with PBS, followed by the addition of 0.3% Triton-100 to induce cell membrane disruption for 20 min. Subsequently, the NK cells were subjected to three washes of 5 min each with PBS. Following this, a concentration of 10 μg/mL phalloidin, known for its strong affinity to filamentous actin (F-actin), was added and incubated overnight at 4 °C Ultimately, the NK cells were stained with DAPI for 10 min, followed by three additional washes with PBS. Fluorescence imaging was performed using Zeiss ZEN 2.3 blue imaging software (Carl Zeiss, Oberkochen, Germany).

The tumor tissues from 4T-1 cell-bearing mice were fixed in 4% paraformaldehyde at 4 °C overnight. Dehydration was carried out by incubation in a sucrose gradient, 5% sucrose for 1 h, 10% sucrose for 2 h, 20% sucrose overnight and 30% sucrose overnight until the tumor tissues completely bottomed. The dehydrated tumor tissues were embedded in OCT (SAKURA, Shanghai, China, Cat# 4583), snap-frozen in liquid nitrogen and stored at −80 °C. The embedded tumor tissues were cut into 10 μm continuous sections using a frozen microtome (Leica Biosystems, Nußloch, Germany) and pasted on adhesive slides. After one night, tumor tissues were washed 3 times with PBS for 5 min each time. Slides were blocked for 30 min at RT with 10% donkey serum diluted in PBS and incubated overnight at 4 °C with FITC-labeled anti-CD49b antibody. After three washed with PBS, the slides were stained with DAPI for 10 min. Finally, after three washed with PBS, the slides were sealed with an anti-fluorescence quenching sealing solution. Fluorescence imaging was performed and processed by Zeiss ZEN 2.3 Blue imaging software (Carl Zeiss, Oberkochen, Germany).