Image it fix perm kit

Intracellular SREBP-1 staining was performed using Image-iT® Fix Perm Kit (Thermo Fisher Scientific Inc.). Briefly, cells were fixed and permeabilized in Fixation Buffer and Permeabilization Buffer for 15 min, respectively. The cells were blocked and incubated with a primary monoclonal antibody against SREBP-1 (BioVision Inc., 1:100 dilution). After incubation with fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific Inc.), cells were counterstained with DAPI (Invitrogen, Thermo Fisher Scientific Inc). After washing with PBS, cells were examined using an FV10i confocal laser microscope (Olympus) or the ImageXpress^® Micro XLS Widefield High-Content Analysis System (Molecular Devices). Data analysis was performed using the MetaXpress software package (Molecular Devices).

Cells were seeded at a density of 100,000 cells per ml in an open μ-Slide (ibid, 80826), and then transfected with siRNAs. After 72 hours, cells were fixed/permeabilized using an Image-IT FIX-perm kit (Thermo Fisher Scientific, R37602) and incubated with an antibody against ALDH (1:100; BD, 611194). The primary antibody was detected with Alexa Fluor 488 goat anti-mouse (1:100; Thermo Fisher Scientific, T7458). DAPI was used for staining the nuclei. After that, confocal observation and quantification of expression were performed using the ZEN LSM 800 and the Zen 2.3 lite program (Carl Zeiss), respectively. Briefly, once confocal observation was performed: at least five frames for each sample were obtained. To quantify immunofluorescence of target proteins, we used a Zen 2.3 lite program (Carl Zeiss) as follows: regions of interest (ROI) were defined using the Histo tool, thereby each group contained an equal numbers of cells. To do that, the integrated density value (IDV) was assessed for the blue channel (DAPI staining). Then, the IDVs were determined for all target proteins provided in the Histo tool. After that, each IDVs were divided by the mean IDV value. Each value was given a log 2 for global normalization.

THP-1 cells, macrophages derived from the bone marrow, or SILP cells were plated
overnight in a 96 well glass bottom plate in DMEM with 10% FBS. This was
followed by 24 h by a pretreatment of LPS at 5 µg/ml, and then followed by 24 h
of nigericin at 5 µg/ml with an without MCC950 at 1 µM. Cells were fixed and
permeabilized using the Image-It Fix-Perm kit (life Technologies). Primary Abs
were added at 1:500: rabbit anti-ASC/TMS1 (Sigma S1B8731) and goat
anti-NLRP3/CIAS1 (Sigma SAB2501362) and incubated for 2 h at 37°C. The PLA
probes were added at 1:5: PLA probe anti-goat minus (DUO92006) and PLA probe
anti-rabbit plus (Duo92002) for 1 h at 37°C. Ligation buffer ligase was added at
1:40 and added to cells for 30 min at 37°C. Amplification buffer polymerase was
added at 1:80 and added to cells for 100 min at 37°C. Cells were washed and
mounted with DAPI (4′,6-diamidino-2-phenylindole). Immune fluorescence staining
was analyzed on an Opera high content screening microscope.

PDL specimens were fixed, permeabilized, and blocked following the Image-iT Fix-Perm kit (Life Technologies, Carlsbad, CA, USA) recommendations. NucBlue Fixed Cell ReadyProbes (Life Technologies) were used for staining the nuclei. Immunoreactivity was visualized with a confocal microscope (Zeiss LSM 700) combined with the Zen software (Carl Zeiss, New York, NY, USA).