Prolong gold antifade mountant with dapi

Synovial lining macrophages were quantified in liposome-treated joint tissue sections (n = 3 per condition) using immunofluorescence labelling as previously described [7 (link)]. Sections were labeled with rabbit anti-CD68 (Abcam) or rabbit polyclonal isotype control (Abcam, ab37415) at 0.005 mg/mL in blocking buffer overnight at 4^oC. Goat anti-rabbit-Alexa-488 secondary antibody (Jackson ImmunoResearch) at 0.0015 mg/mL was applied before washing and mounting with ProLong Gold Antifade Mountant with DAPI (Fisher Scientific). Slides were imaged on a Zeiss LSM 800 AiryScan confocal microscope using the 40X water immersion lens with configuration settings held constant across all samples. Two regions of interest were imaged per section.

Cells were prepared for immunocytochemistry (ICC) by initial fixation using 4.0% formaldehyde (prepared from a 37% stock; Millipore Sigma) in 1.0 M phosphate-buffer saline (PBS), pH 7.4, for 15 min at room temperature. After fixation, and three washes with PBS, cells were permeabilized with 0.3% Triton X-100 (Millipore Sigma, Mississauga, ON, Canada)/PBS for 30 min. Cells were washed three times with PBS and then blocked with Sea Block Blocking Buffer (Thermo Fisher Scientific) for 60 min at room temperature, blocking for non-specific antibody sites. Primary antibodies (Table 1) were diluted in 0.1% Triton X-100/PBS with 10% Sea Block and incubated overnight at 4 °C. Cells were then washed three times with PBS before incubation with secondary antibodies diluted in 0.1% Triton X-100/PBS with 5% Sea Block (Table 1) for 60 min at room temperature. Cells were then washed three times with PBS and mounted in ProLong Gold Antifade Mountant with DAPI (Fisher Scientific). All images and quantification are representative of three or more biological replicates. Random fields of view (RFV; ~5–6 per experiment) were acquired for quantification, and ~50–60 cells were analyzed per experiment. All images were acquired using identical settings within each experiment.

Cells were prepared for immunocytochemistry (ICC) by initial fixation using 4.0% formaldehyde (prepared from a 37% stock; Millipore Sigma) in 1.0M phosphate-buffer saline (PBS), pH 7.4, for 15 min at room temperature. After fixation, and three washes with PBS, cells were permeabilized with 0.3% Triton X-100 (Millipore Sigma)/PBS for 30 min. Cells were washed three times with PBS and then blocked with Sea Block Blocking Buffer (ThermoFisher Scientific) for 1 hour at room temperature. Primary antibodies (Key Resources Table) were diluted in 0.1% Triton X‐100/PBS with 10% Sea Block and incubated overnight at 4°C. Cells were then washed three times with PBS before incubation with secondary antibodies diluted in 0.1% Triton X‐100/PBS with 5% Sea Block (Key Resources Table) for 1 hour at room temperature. Cells were then washed three times with PBS and mounted in ProLong Gold Antifade Mountant with DAPI (Fisher Scientific). All images and quantification are representative of three biological replicates. 40X random fields of view (Plan Apochromat ×40 Oil objective, with a 1.40 numerical aperture; 5 per experiment) were acquired for quantification, and 50 cells were analyzed per experiment. All images were acquired using identical settings within each experiment.

De‐paraffinized sections following antigen retrieval were blocked in 5% normal serum in PBS‐T and incubated with antibodies against collagen I (1:100, Abcam ab34710), collagen II (1:400, Fitzgerald 70R‐CR008), collagen X (1:500, Abcam ab58632), aggrecan (1:50, Millipore AB1031); chondroitin sulphate (1:300, Abcam ab11570); CA3 (1:150, SantaCruz), Ki67 (1:100, Abcam ab15580) and p21 (1:200, Novus NB100‐1941). For GLUT‐1 (1:200, Abcam, ab40084) and ARGxx (1:200, Abcam, ab3773) staining, MOM kit (Vector laboratories, BMK‐2202) was used for blocking and primary antibody incubation. Tissue sections were washed and incubated with Alexa Fluor‐594‐conjugated secondary antibody (Jackson ImmunoResearch Lab, Inc., 1:700). The sections were mounted with ProLong^® Gold Antifade Mountant with DAPI (Fisher Scientific, P36934) and visualized with Axio Imager 2 microscope using 5×/0.15 N‐Achroplan or 20×/0.5 EC Plan‐Neofluar objectives (Carl Zeiss). Staining area and cell number quantification was performed using the ImageJ software. Images were thresholder to subtract the background, transformed into binary, and then staining area and cell number were calculated using the analyse particles function.

Tumors were harvested 2 weeks after the beginning of treatments and were formalin-fixed and paraffin-embedded. Four micrometer thick paraffin-embedded tissue sections were cut and immunostained using an automated Ventana Discovery XT staining system (Ventana Medical Systems). Antigen retrieval was performed in cell conditioner 1 and slides were incubated with anti-phospho-histone γ-H2AX antibody (1:250 dilutions in PBS at 37 °C for 60 min, clone JBW301, EMD Millipore, Temecula, CA) on automatic. Then, on the bench, slides were incubated for 20 min with blocking solution (Dako, Agilent, #X0909) followed by incubation with the secondary antibody (1:250 dilution in PBS at RT for 45 min; anti-mouse Cy5, Life Technologies Inc., #A10524). Finally, slides were incubated 15 min at RT with a 0.1% (w/v) solution of Sudan Black in 70% ethanol to quench tissue auto fluorescence. Slides were mounted using ProLong^® Gold Antifade Mountant with DAPI (Life Technologies Inc., P-36931). Between each step, except after the blocking steps, slides were washed twice with PBS. Slides were stored at 4 °C and scanned the next day. A negative control slide was done in parallel where PBS replaced the primary antibody.