Dp70 microscope

Lung tissues were fixed first in 10% formalin, followed by embedding in paraffin. Five-micrometer-thick (5 μm) slices were stained with hematoxylin-eosin and examined under an Olympus DP70 microscope (Olympus Corporation). A second set were used for immunohistology, using rabbit anti-influenza A virus nucleoprotein (NP) antibody (Serotech). Briefly, tissue sections were de-paraffinized and then fixed with 100% chilled-acetone for 2 h. The sections were incubated with 3% H₂O₂ for 15 min at 37°C before blocking with 5% bovine serum albumin (dilution in PBS) for 1 h. The blocked sections were then incubated with rabbit anti-influenza A virus NP antibody (1:1000 dilution) at room temperature for 1 h. The labeled tissue sections were stained with horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (Sigma-Aldrich) and alkaline phosphatase substrate, and then counterstained with hematoxylin QS (Vector Laboratories).

Immunoreactivity for TRPV1 and TRPA1 in lung tissues was measured using immunohistochemistry. Briefly, after dewaxing and rehydrating, sections (5 μm thickness) were incubated with 3% H₂O₂ for blocking endogenous peroxidase, and then treated with EDTA (pH 8.0) for 3 min in high pressure cooker for antigen retrieve. To reduce nonspecific binding, sections were incubated with 10% normal goat serum for 20 min at 37 °C. Sections were then incubated with primary rabbit antibodies against TRPV1 (1:2200) and TRPA1 (1:3500) at 4 °C overnight. After washing for 3 times with PBS, sections were incubated with poly-HRP anti-rabbit IgG secondary antibody (1:1000) for 20 min. Positive signals were developed using DAB solution. The stained tissue sections were counterstained with hematoxylin. Immunoreactivity for TRPV1 and TRPA1 was visualized under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan) and scanned using NanozoomerS210 whole slides scanner (Hamamatsu Photonics, Hamamatsu City, Japan). Six visual fields were chosen randomly per guinea pig, and images were analysed with Image-Pro Plus 6.0 software.

Paraffin sections were deparaffinized in xylene and rehydrated in a series of graded alcohols, while endogenous peroxidase was quenched with 3% hydrogen peroxide (H₂O₂) for 10 min. For antigen retrieval, sections were placed in citrate buffer (pH = 6), microwaved (Sharp, R-331ZX) for 3 min at 95 °C, and allowed to cool down for 20 min at 26 °C; this process was repeated once. After blocking with a blocking reagent for 30 min at room temperature, sections were incubated with primary antibodies against SP (1:5000), NK1R (1:2500), and CGRP, (1:3000) overnight at 4 °C. After washing, sections were then incubated with poly-horseradish peroxidase (HRP) anti-rabbit IgG secondary antibodies (1:1000) or poly HRP anti-mouse IgG secondary antibodies (1:1000) for 20 min. Immunoreactivity was developed with DAB (3,3′-Diaminobenzidine tetrahydrochloride, a substrate of HRP) for 20 min. After washing, sections were counterstained with hematoxylin.
Immunoreactivity for SP, NK1R, and CGRP was visualized and quantified using an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan) and a NanozoomerS210 whole slide scanner (Hamamatsu Photonics, Hamamatsu City, Japan). A total of six visual fields in sections per animal were chosen randomly and images were analyzed using Image-Pro Plus 6.0 software.

The expression of GSDMD in isolated AMs was estimated using an immunofluorometric assay. AMs (1 × 10⁶) were cultured on glass coverslips for 24 h, fixed in 4% paraformaldehyde for at least 10 min, and washed three times with PBS. After permeabilization with 0.5% Triton X-100, cells were blocked with 5% BSA (60 min) and incubated overnight on a shaker at 4 °C with rabbit polyclonal anti-GSDMD antibody (1:200; catalog no. NBP2-33422, Novus Biologicals, USA). The sections were then incubated in the dark at room temperature for 1 h with donkey anti-goat IgG antibody conjugated with Alexa Fluor 488 (1:200; Invitrogen, USA). Finally, cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, China), then viewed under a fluorescence microscope (Olympus BX51).
Image quantification was performed on three randomly selected, non-overlapping fields per section (three sections per mouse) at 100× magnification using an Olympus DP70 microscope (Tokyo, Japan), and the number of pixels per image with an intensity above a predetermined threshold level was quantified using ImageJ software (version 1.47n; National Institutes of Health, Bethesda, MD, USA). The immunoreactivity was expressed as the percentage of total pixels of the imaged field that had an intensity above the threshold level. All quantitative analyses were performed in a blinded manner.