Alexa fluor conjugated secondary antibody

Reagents were obtained from Sigma Aldrich (St. Louis, MO) or WAKO (Osaka, Japan) unless otherwise indicated. Triton X-100 was purchased from MP Biomedicals (Aurora, OH). Fetal bovine serum was obtained from Biosera (Biosera, Nuaillé, Chile). Collagenase type IV was purchased from Worthington (Lakewood, NJ). H₂DCF-DA (2',7'-dichlorodihydrofluorescein diacetate), JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide), ProLong Diamond Antifade Mountant with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), Alexa Fluor 488-conjugated phalloidin and MitoTracker Red (CMXRos) were from Molecular Probes (Eugene, OR). Alexa Fluor-conjugated secondary antibodies were from Cell Signaling (Danvers, MA). Anti-α-actinin antibodies were from Sigma (A7811). All reagents from commercial sources were of analytical grade.

All chemicals were of analytical grade or higher. Primary antibodies and AlexaFluor^®-conjugated secondary antibodies were purchased from Cell Signaling Technology (Leiden, The Netherlands) or ThermoFisher (ThermoFisher Scientific, Darmstadt, Germany). IRDye^®-conjugated secondary antibodies were purchased from LI-COR Biosciences (Bad Homburg, Germany). A full list of used antibodies and dilutions is provided in Supplementary Table S1.

Paraffin-embedded sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome (MT) or Sirius red (SR). For lipid staining, frozen sections were stained with oil red O and counterstained with hematoxylin. The NAFLD activity score and fibrosis stage were scored by an outsourcing company (Allisere, Tokyo, Japan) in a blind manner according to the method of Kleiner et al. (S1 Table) [27 (link)]. Immunohistochemistry was carried out on cryostat liver sections (thickness, 7 μm). Sections were incubated with primary antibodies and stained with Alexa Fluor-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Apoptosis was assessed by TUNEL staining of paraffin-embedded slides. Images of five random fields of each section were selected and the number of apoptotic cells per field was counted. To quantify the area of SR staining and α-smooth muscle actin (α-SMA) staining, images of five random fields of each section were processed with Photoshop Elements v13 (Adobe Systems, San Jose, CA, USA). Each value was expressed as the percentage of the total area of the section.

RAW264.7 macrophages were fixed with 4% paraformaldehyde for 15 min and permeated with 0.3% Triton X-100 for 15 min at room temperature. After washed with PBS for 1 h, the cells were incubated with primary antibodies against hnRNPK, FLIP and NLRP3 (Abcam) at 4 °C overnight. The coverslips were exposed to Alexa Fluor conjugated-secondary antibodies (Cell Signaling Technology) for 1 h in the dark. The nucleus was marked with 4′, 6-Diamidino-2-phenylindole (DAPI). The stained images were observed using an Inverted/Fluorescence Microscope (LIONHEART ^LX, BioTek).

Immunofluorescence staining was performed on 10% v/v formalin fixed, paraffin embedded left colon biopsies. Sections were deparaffinized and subjected to 0.01 M citrate buffer (pH 6.0) antigen retrieval at 121 °C for 4 min in a decloaking chamber. Non-specific binding was blocked by a 1-h incubation in blocking buffer [0.1 mol/L phosphate-buffered saline (PBS)/5% normal goat serum/0.05% Tween-20] at room temperature in the dark. Immunofluorescence staining was performed using specific antibodies against NLRP3 (ab16097, Abcam, Cambridge, MA, USA) and IL-1β (ab9722, Abcam, Cambridge, MA, USA) in the dark, at room temperature for 1 h or alternatively overnight at 4 °C. Sections were then incubated for 1 h in the dark with one or more Alexa Fluor conjugated-secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Section were incubated with 4′,6-diamidino-2-phenylindole Dihydrochloride (DAPI, ThermoFisher Scientific, Waltham, MA, USA) diluted in PBS and mounted with ProLong Gold Antifade (P36930, ThermoFisher Scientific, Waltham, MA, USA). Slides were examined using a FV1200 Laser Scanning Confocal Inverted Microscope (Olympus Australia, Melbourne, Australia).