Ab55152

The cells were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% triton X-100 in PBS for 15 min. After washing with PBS buffer three times, cells were blocked in 1% BSA in TBST solution for 1 h and then incubated with diluted MHC primary antibody (Rabbit anti-MHC class I, ab281901, Abcam, 1:100, and mouse anti-MHC class II, ab55152, Abcam, 1:100) at 4˚C for overnight. After washing with PBS, cells were incubated second antibodies conjugated with Alexa Fluor 488 and 594. (Goat anti rabbit Alexa Fluor 488, ab150081, 1:500 and Goat anti-mouse Alexa Fluor 594, A-11032, Thermo Fisher Scientific, 1:200) for 1 h at room temperature. 1 µg/ml of Hoechst 33342 was used for the staining of nuclei acid. All images were taken using a fluorescent microscope (Nikon Eclipse Ts2) equipped with 10× objective lens (Plan Fluor, Nikon) and image software (NIS element version 4.51.00, Nikon). For the image analysis, 2–3 randomly selected fields were used from three independent experiments. Evaluation of fluorescence intensity was carried out with ImageJ FIJI software^{55 (link)}.

LCs were separated from the PBMCs of patients in the different groups as previously described [24 (link)]. Briefly, PBMCs were incubated in RPMI medium supplemented with 1000 U/ml GM-CSF, 2 mM L-glutamine, 10 mg of streptomycin, 10% foetal bovine serum (FBS), 1000 U/ml IL-4, and 10 μg/ml TGF-β for 7 days. The LC phenotype (CD1a + Langerin + CD14-) was confirmed by flow cytometry.
Surface marker expression was analysed by flow cytometry. Briefly, 10⁶ LCs were harvested, washed, stained with antibodies against MHC I (ab134189, Abcam), MHC II (ab55152, Abcam), CD80 (ab134120, Abcam), CD83 (ab275021, Abcam), CD86 (ab239075, Abcam), and CD40 (ab224639, Abcam Inc., Cambridge, UK) or with isotype controls for 1 h, and washed with FACS buffer. All the flow cytometric analyses were performed using an FC500 flow cytometer. Then, we treated the LCs with 20 μM LY294002 (EMD Biosciences), a potent specific inhibitor of PI3K, and analysed surface marker expression again. The flow cytometric data were quantified as the median fluorescence intensity (MFI) and analysed using FlowJo software (FlowJo, LLC).

Fresh tissue sections were obtained from the subcutaneous obese AT, they were placed in O.C.T. blocks (Tissue-Tek 4583) and stored at −80°C. Blocks were sectioned at 16 μm thickness and fixed in cold acetone for 10 min. For the staining, the tissue sections were fixed in acetone and washed twice with PBS. The tissue sections were then blocked for 1 h with 5% BSA at room temperature, and incubated with primary anti-CD1d antibody (abcam ab11076), or with primary anti-MHC class II antibody (abcam ab55152), both 1:100 diluted, overnight at 4°C. The next day, the slides were washed with PBS twice for 5 min and incubated with the following secondary antibodies: AF488-conjugated goat anti-mouse IgG (Biolegend 405319) for CD1d detection, and AF647-conjugated goat anti-mouse IgG (ThermoFisher InVitrogen A-21236) for MHC class II detection, both 1:100 diluted, 1 h at room temperature, or with the isotype controls [IgG1 (Biolegend 401402) and IgG2a (Biolegend 401502), respectively]. The slides where then washed 3 times, 3 min each. The cover slides were mounted with ProLong Gold Antifade Mountant and DAPI (4′,6-diamidino-2-phenylindole, ThermoFisher Scientific), which stains the nuclei of immune cells, to visualize the nuclei. Slides were imaged with a Keyence inverted microscope.