Ab53082

Paraffin-embedded samples were consecutively cut to a thickness of 4–5 μm and placed on SuperFrost plus slides (Thermo Scientific, Waltham, MA, USA). Each section was deparaffinized and rehydrated with graded ethanol. Antigen retrieval was performed by microwave heating for 20 min in 10 mM sodium citrate buffer (pH 6.0). The activity of endogenous peroxidase was blocked with a 3% hydrogen peroxide solution (Abcam, Cambridge, UK) for 10 min at room temperature. After washing them with PBS, the slides were incubated with primary mono/polyclonal antibodies against NLRP3 (ab214185, Abcam, Cambridge, UK, at a 1:500 dilution), IL-1β (ab9722, Abcam, Cambridge, UK, at 1 μg/mL), and galectin-3 (ab53082, Abcam, Cambridge, UK, at a 1:100 dilution) overnight in a humid chamber at 4 °C. Antibody-treated sections were washed and stained using rabbit-specific HRP/AEC and HRP/DAB kits (Abcam, Cambridge, UK) according to the manufacturer’s recommendations. For negative controls, identical protocol for immunohistochemistry was performed, but primary antibody was omitted (Figure 1). All of the sections were counterstained in Mayer’s hematoxylin, dehydrated with alcohol, and mounted. An Olympus microscope (BX50 model) equipped with a digital camera was used to prepare microphotographs with magnifications of 100× or 400×.

Formalin-fixed, paraffin-embedded mouse liver tissue sections were incubated with rabbit anti-TNF-α (ab66579, Abcam), rabbit anti-caspase-3, active/cleaved (NB100-56113, Novus Biologicals), anti-caspase 3 and rabbit anti-Gal-3 (ab53082, Abcam). Sections were visualized by rabbit-specific conjugate (Expose Mouse and RabbitSpecific HRP/DAB Detection IHC Kit; Abcam) and photomicrographed with a digital camera mounted on light microscope (BX51; Olympus). Virus-infected cells were visualized by anti-IE1 staining (MCMV protein expressed with early kinetics).

For immunohistochemical staining, we used paraffin-embedded sections (5 μm) of the mouse pancreas tissue. Deparaffinized tissue sections were incubated with primary mouse anti-Galectin 3 antibody (ab53082, Abcam), anti-Caspase/3 antibody (ab184787, Abcam), anti-Ki-67 (ab16667, Abcam), or insulin antibody (ab63820, Abcam). Staining was visualized by using the mouse-specific HRP/DAB detection IHC kit (ab64259, Abcam), and the sections were counterstained with Mayer’s hematoxylin. The tissue slices were not consecutive, and the analyzed islets were randomly chosen for each parameter. In each analysis, islets with different insulitis score were randomly included. The sections were photomicrographed with a digital camera mounted on a light microscope (Olympus BX51, Japan), digitized, and analyzed. Quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), on 10 fields/section. Scoring and histological analysis were performed in blinded fashion by two independent observers. Results are presented as a mean count of positive cells per islet area or the percentage of islet area.

A representative FFPE section from each tissue block (5 μm thickness) was deparaffinized, hydrated with series of graded alcohol and then antigen retrieval at 115 °C for 3 min was performed in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) as described previously [40 (link)]. Tissues were treated with 3% H₂O₂ in Tris-buffered saline for 8 min to block the endogenous peroxidase activity. Then slides were incubated with a background punisher to block non-specific staining (Biocare Medical, LLC, Concord, CA, USA) for 20 min, and were followed by 1 hr incubation with rabbit anti-Gal-3 antibody at a 1:500 dilution (ab53082, Abcam, Cambridge, MA, USA). The rinsed sections were then incubated with biotinylated anti-rabbit secondary antibody for 20 min, subsequently detected using The VECTASTAIN ABC System (Vector Labs, Burlington, ON, Canada) and peroxidase substrate diaminobenzidine (DAB) as the chromogen until staining signal developed. Sections of a classical variant of PTC known with Gal-3 positivity were included in each batch of immunostaining for Gal-3 antibody incubation to serve as an external positive control as well as for an irrelevant isotype specific IgG in place of the primary antibody as a negative control to exclude a nonspecific staining. All slides were counterstained with hematoxylin and viewed under a light microscope.

The antibodies used for immunohistochemistry staining and immunofluorescence staining were purchased from Abcam (Shanghai, China) including anti-TNF-α antibody (ab6671, 1:200), anti-IL-1β antibody (ab9722, 1:400), anti-IL-6 antibody (ab83339, 1:2000), anti-F4/80 antibody (ab6640, for immunohistochemistry staining, 1:200), anti-F4/80 antibody (Alexa Fluor 488, ab204266, for immunofluorescence staining, 1:400), anti-inducible nitric oxide synthase (iNOS) antibody (Alexa Fluor 647, ab209027, 1:200), anti-Mannose receptor antibody (anti-CD206, Alexa Fluor 647, ab195192, 1:200), anti-Galectin 3 (Mac-2) antibody (ab53082, 1:200).