Silane coated slides

For immunohistochemical studies, the tumor, spleen, liver, BM, lung, skin, gut, and kidney of HSC-NOG-hIL-6 Tg mice or HSC-NOG non-Tg mice were fixed in Mildform 10MN formaldehyde solution (Wako Pure Chemical, Osaka, Japan) and embedded in paraffin. The samples were serially sectioned into 3-µm thicknesses using a microtome, and placed on silane-coated slides (Muto Pure Chemicals, Tokyo, Japan). Immunostaining was performed using a Leica Bond-Max automatic immunostainer (Leica Biosystems, Mount Waverley, VIC, Australia). Paraffin sections were dewaxed in a Bond Dewax solution and rehydrated in alcohol and Bond Wash solution (Leica Biosystems). Antigen retrieval was performed using a retrieval solution (ER1, 10 mM citrate buffer, pH 6), followed by endogenous peroxidase blocking. Detection was performed using a Bond Polymer Refine Detection system. Then, the sections were counterstained with hematoxylin. We used monoclonal anti-human CD68 (clone: PG-M1, DakoCytomation, Glostrup, Denmark) and monoclonal anti-human CD163 (clone: 10D6, Leica Biosystems Newcastle Ltd., Newcastle, UK) antibodies for immunohistochemical analyses (National Institutes of Health, Bethesda, MD, USA).

All samples were examined by IHC with the novel EPIYA-C and EPIYA-D mAbs and the commercially available CagA mAb and H. pylori mAb (D369-3, MBL, Nagoya, Japan) specific to H. pylori lipopolysaccharide (LPS). Serial tissue sections (4 μm thick) mounted on silane-coated slides (Muto Pure Chemicals Co. Ltd., Tokyo, Japan) were subjected to automated IHC with the Leica BOND-III (Leica Microsystems Inc., Tokyo, Japan) using a BOND Polymer Refine Detection kit (#DS9800, Leica Microsystems Inc.). Deparaffinization, peroxidase inhibition, antigen retrieval, incubation with primary antibody at room temperature for 15 min, and counterstaining were performed according to the manufacturer's protocol. The best protocol for antigen retrieval was determined, as shown in Table 1. Appropriate dilution in Primary Antibody Diluent (10–0001RUO, Sakura Finetek Japan Co., Ltd., Tokyo, Japan) was determined for the H. pylori mAb; 1 : 1000, CagA mAb; 1 : 100, EPIYA-C mAb; 1 : 2000, and EPIYA-D mAb; 1 : 400. The IHC results were considered to be positive when at least 1 cluster of unequivocal dot-like signals was observed on the surface of the gastric mucosa in the biopsy sample.

Ankle joint tissue sections were attached to silane-coated slides (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), baked, and de-paraffinized. Antigen retrieval was performed in a citrate buffer (pH 6.0) overnight at 60 °C. Sections were blocked for 30 min with 1.5% bovine serum albumin (Sigma, St. Louis, MO, USA), followed by incubation with primary antibody against IL-6 (R&D Systems, Minneapolis, CA, USA), IL-1β (R&D Systems), TNF-α (R&D Systems), RANK (R&D Systems), RANKL (R&D Systems), IL-6R (Invitrogen, Waltham, MA, USA), or TNF-αR (Invitrogen) overnight at 4 °C (Supplementary Table S2). Biotinylated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used to detect primary antibodies. Thereafter, a streptavidin-tagged horseradish peroxidase kit (Vector Laboratories, Burlingame, CA, USA) was used to amplify the signal, and visualization of the antibody was performed using a NovaRED detection kit (Vector Laboratories, Burlingame, CA, USA). Mayer’s hematoxylin (BBC Biochemical, Stanwood, WA, USA) was used as a counterstain. Stained slides were photographed by optical microscopy (Leica, Wetzlar, Germany) and rendered using Leica software. A light microscope was used for image processing, and immunohistochemistry signals were quantified using ImageJ software (Version 1.53, NIH, Bethesda, MD, USA).

After fixing in 10% neutral-buffered formalin, bone–tendon–muscle units were decalcified in a decalcifying solution (TBD-1 decalcifier, Thermo Scientific, Kalamazoo, MI, USA). The decalcification process was terminated when the bone could be penetrated using a needle without any force. Subsequently, specimens were embedded in paraffin, and 4-µm sections were cut and placed on silane-coated slides (Muto Pure Chemicals, Tokyo, Japan). The deparaffinized sections were stained with hematoxylin-eosin and Masson’s trichrome stain. These sections were then examined by light microscopy to assess the morphology of tenocytes, cellularity, and structure or arrangement of collagen fibers.

For histological assessment, the hamsters were euthanized by inhalation of carbon dioxide four days post-infection (dpi). Whole heads of the hamsters were dissected. The mandible, scalp, muscles, calvaria, and brain were removed to facilitate penetration of the fixative, and the rest of the heads were fixed in 10% neutral-buffered formalin for three days at 4 °C. After fixation, they were decalcified in MoL-decalcifier solution (Milestone Medical, Sorisole, BG, Italy) for three weeks at room temperature (25 °C) with gentle shaking. The nasal cavity was then cut into four equal parts, which were processed, embedded in paraffin blocks, cut into 5 µm thick sections, and attached to silane-coated slides (MUTO Pure Chemicals, Tokyo, Japan).
Alcian blue staining was performed using the Alcian blue pH 2.5 staining kit (BBC Biochemical, Mt Vernon, WA, USA). In brief, sections were deparaffinized and rehydrated in xylene and graded alcohols. Sections were stained with Alcian blue solution for 15 min in a 37 °C water bath. After washing, the sections were counterstained with nuclear fast red for 5 min. Sections were dehydrated and cleared in graded alcohol and xylene. Finally, the sections were mounted with a toluene-based mounting medium (Thermo Scientific, Waltham, MA, USA).

IC filter papers with the detached epithelial cells were firmly pressed to silane-coated slides (Muto Pure Chemicals co., ltd. Tokyo, Japan), and epithelial cells transferred to the slides were used for immunofluorescent staining according to Baudouin’s protocol^{50 (link)}. Briefly, cold methanol was used to fix the cells and 0.1% Triton X-100 was applied for cell permeabilization. Then, nonspecific reactions were blocked by incubation with 10% goat serum for 1 h. After, they were then incubated with anti- LC3B-I, II or ATG5 antibody (1:400) and washed twice with PBS. Finally they were incubated with goat Alexa Flour 488–conjugated anti-rabbit IgG Ab (1:400, ThermoFisher scientific, Waltham, MA, USA) in PBS and the stain was captured by confocal microscopy (LSM 510 Meta (Carl Zeiss Meditec Inc. Dublin, CA) and transferred to Photoshop (Adobe systems, Santa Clara, CA).