Gold antifade mountant with dapi

A549 and 293FT cells were grown on coverslips and transfected with the indicated plasmid. At 24 h after transfection, cells were washed once with PBS and fixed in 4% paraformaldehyde in PBS. Subsequently, cells were permeabilized with 0.2% Triton X-100 and treated for 30 min at room temperature with 10% BSA in PBS, followed by incubation with primary antibody for 1 h. Primary anti-PHF14 antibody (Proteintech, 24787-1-AP) was used. The secondary antibody was goat anti-rabbit IgG (H + L) conjugated with Alexa Fluor 594 (Thermo Fisher Scientific) in 1% BSA at room temperature for 20–90 min. The cells were rinsed 2–3 times with PBS (pH7.4) (5 min/wash) and then mounted with Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). The coverslips were washed extensively and fixed on slides, and representative images were obtained with a LSM810 confocal microscope using ZEN 2012 software version 8.1 (Carl Zeiss, Oberkochen, Germany).

HD11 cells or primary monocytes grown on glass coverslips were stimulated with 10 μg/ml cNK-2 or medium alone for 30 min. The cells were fixed in 4% paraformaldehyde (Sigma) for 10 min, permeabilized in 0.1% Triton X-100 in PBS for 15 min, blocked in SuperBlock (PBS) Blocking Buffer (Thermo Fisher Scientific) containing 2% normal goat serum for 1 h, and incubated with 10 μg/ml cNK-2 rabbit polyclonal antibody for 1 h at room temperature. After washing three times with PBS-T, the cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibodies (Thermo Fisher Scientific) for 1 h. After repeated washing with 0.1% Tween 20 in PBS, the cells were stained with Alexa Fluor 555 Phalloidin (Cell Signaling), mounted using Gold Antifade Mountant with DAPI (Thermo Fisher Scientific), and examined using an Eclipse 80i fluorescence microscope (Nikon, Japan), using a fixed shutter speed to permit fluorescence intensity comparison. For confocal microscopic images, a Zeiss 710 confocal laser scanning microscopy (CLSM) system was utilized.

Adipose tissues were fixed in 4% formalin PBS solution for 24 hrs, dehydrated, embedded in paraffin and sectioned at 5 μm. The sections were deparaffinized, re-hydrated and then subjected to haematoxylin and eosin staining (Sigma) or immunofluorescence staining. For immune-staining, the sections were blocked in 3% BSA (Roche), 10% FBS (Gibco), PBS, pH = 7.4 for 1 hr, followed by incubation with primary antibodies in PBST (PBS, 0.1% Tween 20) containing 3% BSA overnight at 4 °C. The next day, the slides were washed three times in PBST followed by incubation with a fluorophore-conjugated secondary antibody for 1 hr at dark. Slides were counterstained with Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and visualized with an Olympus biological microscope BX41, and images were captured with an Olympus DP72 color digital camera (for HE staining) and Carl Zeiss LSM800 inverted confocal microscope (immunofluorescence staining). The antibodies used include F4/80 (1:100, Thermo Fisher Scientific, #14-4801-85, BM8), iNOS (1:250, Thermo Fisher Scientific, #PA3-030A), HIF-1α (1:400, Proteintech, 20960-1-AP), CD14 (1:100, Biolegend, #325604, HCD14), Chicken anti-Rat IgG (1:200, Thermo Fisher Scientific, #A21470) and Goat anti-Rabbit IgG (1:200, Thermo Fisher Scientific, #A11011).