Alexa fluor 488 labeled anti rabbit

Paraffin-embedded samples were sectioned at 4 μm thickness.
Antigen retrieval was performed by a pressure cooker for (95°C for 30
min) in Antigen Unmarking Solution (Vector Laboratories, Cat# H-3300).
Sections were then blocked in PBS containing 2% bovine serum albumin for 1
hr at room temperature. For dual immunofluorescence staining, the slides
were incubated in the mixture of two primary antibodies overnight at 4
^oC. The following primary antibodies were used: rabbit
anti-CXCR2 (Abcam, Cat# ab14935), rat anti-Gr-1 (BioLegend, Cat# 108401),
mouse anti-Cytokeratin Pan (Abcam, Cat# ab7753), rabbit anti-CD4 (Abcam,
Cat# ab183685), rabbit anti-CD8 (Bioss, Cat# bs-0648R), and rat anti-FOXP3
(eBioscience, Cat# 14–4771-80). The slides were washed with cold PBS
and incubated with the mixture of two secondary antibodies which are raised
in different species for 1 hr at room temperature in dark. The following
secondary antibodies were used: Alexa Fluor 488 labeled anti-rabbit (Life
Technologies, Cat# A11008), Alexa Fluor 594 labeled anti-rat (Life
Technologies, Cat# A11007), Alexa Fluor 488 labeled anti-mouse (Life
Technologies, Cat# A11001), and Alexa Fluor 594 labeled anti-mouse (Life
Technologies, Cat# A21203). Slides were counter-stained with DAPI/anti-fade
mounting medium (Vector Laboratories) and examined by fluorescence
microscopy (Leica DFC345 FX).

Twenty four hours after siRNA or Cas9/sgRNA RNP transfection, U2OS cells were seeded on black 96-well bottom-glass plates. After 3 additional days of incubation, cells were simultaneously fixed and permeabilized (4% paraformaldehyde, 0.5% Triton X-100) for 10 min at room temperature. Cells were incubated in blocking solution (3% BSA in TBS-Tween 0.1%) for 1 hr and then with primary antibody diluted in blocking solution for 1.5 hr at room temperature or overnight at 4°C. Cells were washed 3 times with TBS-T and then incubated for 1 hr at room temperature with the appropriate secondary antibody, Alexa Fluor 488-labeled anti-rabbit and Alexa Fluor 594-labeled goat anti-mouse at 1:1,000 dilution (A-11008 and A-11005, Thermo Fisher Scientific). After three washes in TBS-T, cells were incubated with DAPI for 5 min at room temperature to counterstain nuclei. Multi-color acquisitions were made using the ImageXpress Nano Automated Imaging System microscope (Molecular Devices) equipped with a 40× Plan Apo objective (0.95 numerical aperture). An integrated imaging software (MetaXpress 6) was used for image analysis. At least 300 cells per experimental point were analyzed, and each experiment was repeated at least 2 times independently.