Alexa fluor 488 anti mouse f4 80 antibody

Gastrocnemius muscles were embedded in paraffin and cut into 4 μm sections. After blocking 1 h with 5% bovine serum albumin/PBS containing 0.1% Tween 20 at room temperature, sections were incubated with Alexa Fluor 488 anti-mouse F4/80 antibody (1:100 dilution; 123120, BioLegend, San Diego, CA) and anti-TNF alpha antibody (1:100; ab6671, Abcam, Cambridge, UK) overnight at 4 °C. After three PBS washes, sections were stained 1 h with F(ab')2-Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (1:200; Invitrogen, A-11071, Waltham, MA) at room temperature. DAPI (1:5000; Wako Pure Chemicals Industries, Osaka, Japan) was used for a nuclear stain. The number of F4/80/TNFα double-positive cells and DAPI positive nuclei was determined in five randomly selected 500-μm squares using a fluorescence microscope (BioRevo, Keyence, Osaka, Japan).

Eight to ten weeks-old, C57BL/6NJ female mice were exposed by intratracheal intubation with 40 μl aqueous suspensions of 200 μg silica crystals (MIN-U-SIL-15) or 200 μg red-fluorescent silica beads (control mice) in PBS. Mice were euthanised 16–18 h after instillation and bronchoalveolar lavage fluid was obtained by three consecutive instillations and withdrawals of 1 ml of 2 mM EDTA and 0.5% FCS in PBS. Recovered fluid was pelleted by centrifugation and treated with 300 μl of red blood lysis buffer for 5 min on ice. After that, cells were washed with 20 ml of PBS and centrifugated at 400 × g for 7 min. Cells were then resuspended in 2 ml of PBS and counted. Cell viability was measured by trypan blue exclusion assay using a TC20 automated cell counter (Biorad). The percentage of viable cells was between 86 and 91%. Subsequently, cells were stained with Alexa Fluor 488 anti-mouse F4/80 antibody (Biolegend, 123120) and plated in µ-Slide 18 well coverslips (Ibidi, 81816) and processed for downstream applications.