Blocking reagent

Tissue samples were fixed with formalin and embedded in paraffin. Following dewaxing and rehydration, heat-induced epitope retrieval was performed by boiling the specimens in 1/200 diluted ImmunoSaver (Nissin EM, Tokyo, Japan) at 98 °C for 45 min. Endogenous peroxidase was inactivated by treating the specimens with 0.3% H₂O₂ in methanol at room temperature for 30 min. Then, the specimens were treated with 0.1% Triton X-100 for permeabilization. After treatment with a blocking reagent (Nacalai Tesque) at 4 °C for 30 min, the specimens were incubated with primary antibodies, anti-mouse mesothelin (295D, D053-3, MBL, dilution 1:100), at room temperature for 1 h or at 4 °C overnight. The sections were stained using ImPRESS IgG-peroxidase kits (Vector Labs) and a metal-enhanced DAB substrate kit (Life Technologies), according to the supplier's instructions. After counterstaining with haematoxylin, specimens were dehydrated and mounted. For actin staining, mesothelial cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, actin was stained with ActinGreen 488 Ready Probes Reagent (R37110, Molecular Probes). Then, after washing with PBS, the nuclei were counterstained with Hoechst 33342 dye (Dojindo). The stained cells were washed in PBS for observation.

rPLY (1 µg/mL) and varying concentrations of matcha supernatant (20–320 μg/mL) or catechins (0.1, or 1 mg/mL) in PBS were mixed and incubated at 37 °C for 1 h. Then, sheep erythrocytes were diluted with the supernatant to a final concentration of 1% (v/v) and incubated at 37 °C for 1 h. Thereafter, the samples were suspended in a 4 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (200 mM Tris-HCl [pH 6.8], 8% SDS, and 0.02% bromophenol blue) without β-mercaptoethanol and incubated at 50 °C for 10 min. After incubation, protein samples (20 μL each) were separated on a 7.5% SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA) at 80 V for 90 min. The membranes were then incubated with a blocking reagent (Nacalai Tesque, Kyoto, Japan) to block nonspecific binding, incubated with anti-PLY monoclonal antibody clone PLY-4 (1:1000; Abcam, Cambridge, UK), and probed with a horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody (1:3000; Cell Signaling Technology, Danvers, MA, USA). Thereafter, the membranes were treated with HRP substrates (GE Healthcare, Little Chalfont, UK) and imaged using an ImageQuant LAS 4000 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

The supernatant from S. pneumoniae strains were mixed with 2% SDS-sample buffer, heated at 99 °C for 3 min, separated by SDS-PAGE using 12.5% gels (Gellex International, Tokyo, Japan), and transferred to polyvinylidene difluoride membranes (Merck Millipore). The membranes were incubated with blocking reagent (Nacalai Tesque) to block nonspecific binding and probed with the anti-PLY antibody (Abcam) diluted in Tris-buffered saline containing 0.05% Tween 20 (TaKaRa, Shiga, Japan). The membrane was then incubated with a HRP-conjugated secondary antibody (Cell Signalling Technologies, Danvers, MA, USA) in Tris-buffered saline containing 0.05% tween 20. The membrane was treated with HRP substrates (GE Healthcare) and analysed by chemiluminescence detector (Fujifilm, Tokyo, Japan). As a loading control, whole-cell lysates of S. pneumoniae strains prepared through the process of bacterial-supernatant preparation were immunoblotted with an antibody specific for pneumococcal mid-cell-anchored protein Z, which is a key membrane protein involved in bacterial-cell division and morphogenesis55 (link).