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.

MDCK-HA cells were grown on coverslips in 6-well plates and infected with scPR8-RBD-M2 and scPR8-mCherry-M2 viruses for 48 h. After infection, cells were fixed with 4% paraformaldehyde at 4 °C for 20 min and blocked under non-permeabilized and permeabilized conditions with 5% FBS and 0.5% BSA at room temperature for 1 h. Cells were then probed with rabbit anti-RBD or anti-S antibodies (Sino Biological, Beijing, China). Goat anti-rabbit antibodies conjugated with Alexa Fluor^® 488 (Abcam, Cambridge, UK) were used as secondary antibodies. After washing, cells were mounted with Prolong^TM Gold Antifade Mountant with DAPI (Invitrogen™, Carlsbad, CA, USA). The samples were observed by Fluoview^TM FV1000 confocal microscopy (Olympus, Tokyo, Japan).

Immunofluorescence was performed according to method in use in the laboratory [34 (link)]. Cells were fixed with absolute alcohol, washed, and, blocked with 3% bovine serum albumin in PBS for 30 min. The cells were then incubated overnight with anti-Vps34 (1:500; Abcam, Cambrige, UK), anti-ATG14L (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-collagen (1:500;Novus Biologicals, CO, USA) and anti-fibronectin (1:500; abcam) antibodies. The cells were washed with blocking solution for 30 min and then incubated for 1 h with goat anti-rabbit Alexa Fluor^® 594 (IgG) secondary antibody (Life Technologies, CA, USA) diluted in blocking solution. Cells were washed with PBS and then mounted with ProLongTM Gold Antifade Mountant with DAPI (Invitrogen). Cells were observed with the Nikon 80i microscope (Nikon, Tokyo, Japan) under the automatical exposure time for each wavelength or Zeiss LSM 880 with Airyscan confocal microscopy (Zeiss, Jena, Germany) wavelengths of 488 and 592 nm. Images were captured with the DP Controller software.

2–3 × 10⁶ cDC1 from day-11-Flt3L-cultured BM cells were stimulated with or without HKLM-OVA, and cells were washed with PBS for three times and then were fixed with pre-warmed 4% paraformaldehyde for 15 min. Then cells were washed with PBS for three times and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After washed three times with PBS, cells were treated with 100 mM Glycine for 10 min. Cells were rinsed with PBS and then were blocked with PBS containing 5% FBS at room temperature for 30 min. Cells were incubated with primary antibodies at 4 °C overnight. After rinsed by PBS, cells were stained with secondary antibodies at room temperature for 4 to 6 h. After washing with PBS for three times, coverslips were mounted in ProLong^TM Gold Antifade Mountant with DAPI (Invitrogen). Confocal images were obtained by Leica TCS SP8 DLS and Zeiss LSM 900+Airyscan2 confocal microscopes.