Pe anti mouse cd206 mmr

Immunofluorescence was performed on frozen liver sections, as previously described^{38 (link)}. Liver sections were stained with the following primary antibodies: FITC Mouse Anti-iNOS/NOS Type II (1:200; BD Transduction Laboratories™, San Jose, CA, USA), PE anti-mouse CD206 (MMR) (1:200; BioLegend Inc., San Diego, CA, USA), Anti-actin, α-Smooth Muscle antibody (1:300; Sigma, St Louis, MO, USA), Anti-mouse MLKL (1:400; Biorbyt, San Francisco, CA, USA), Anti-MLKL (phospho S345) antibody (1:400; Abcam), Anti-RIPK3 (1:400; Abcam), RIP(D94C12) XP Rabbit mAb (1:400; Cell Signaling Technology, MA, USA), anti-NLRP3 antibody (1:300; Abcam), anti-ASC antibody (1:200, Cell Signaling Technology), and anti-cleaved caspase-1 (1:200; Cell Signaling Technology). For indirect immunofluorescent staining, liver sections were incubated with the following secondary antibodies: PE-conjugated donkey anti-rabbit IgG for p-MLKL, MLKL, RIPK3, and SMA (1:500); Alexa Fluor 488-conjugated donkey anti-rabbit IgG for p-MLKL, MLKL, RIPK3, RIPK1, NLRP3, ASC, and cleaved caspase-1 (1:500). Nikon Inverted Fluorescence Microscope ECLIPSE Ti and NIS-Elements F 3.0 Software (Nikon Corporation, Tokyo, Japan) were applied for image capture.

Freshly harvested pancreatic tissue samples were digested in 0.75 mg/mL collagenase-P (Roche Basel, Switzerland) solution at 37°C for 15 min. Subsequently, tissue was homogenized in gentleMACS™ Dissociators (Miltenyi Biotec, Bergisch Gladbach, Germany) and filtered through 75 μm filter screen with phosphate buffer saline (PBS). Single-cell suspensions were incubated for 15 min at room temperature in PBS with the following antibodies: PE/Cy7 anti-mouse CD45, Alexa Fluor 488 anti-mouse F4/80, Brilliant Violet 421 anti-mouse/human CD11b, PE anti-mouse CD206 (MMR), Alexa Fluor 647 anti-mouse Ly6G and APC anti-mouse CD86 from Biolegend (CA, USA). Isotype-matched controls were included in all experiments. Gating methods of fluorescence-activated cell sorting were programmed as CD45⁺CD11b⁺Ly6G⁺ (for neutrophils), CD45⁺CD11b⁺F4/80⁺CD86⁺ (for M1 macrophages) and CD45⁺CD11b⁺F4/80⁺CD206⁺ (for M2 macrophages). Stained cells were analyzed on an Attune NxT flow cytometer (Thermo Fisher Scientific, MA, USA).