Axio observer z1 microscope

Animals were transcardially perfused with 4% paraformaldehyde in phosphate buffer; brains were harvested, sectioned into 25 μm by cryostat and processed for immunohistochemistry. Brain sections were blocked with 5% normal goat serum and 3% bovine serum albumin in 0.1 M Phosphate Buffer (PB) followed by primary antibody incubation overnight and by corresponding secondary antibodies. (Antibody lists are in Supplementary Table.) Fluorescence images were captured using Tissue Gnostics Axio Observer Z1 microscope (TissueGnostics GmbH, Vienna, Austria) in Fig. 2 and Supplementary Fig. 5. Sections from various groups were reacted in the same well and controls consisted of omission of the primary antibody. Animals were perfused with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PB for transmission electron microscopy; brain tissues including sensory cortex, DG, and CA1 regions were cut out and postfixed with OsO4. After washing and serial dehydration, tissues were treated with PO (propylenoxide) and Epon following by embedding into resin and sectioning into 0.38 μm. Samples were examined and photographed using TEM HT7700 (HITACHI, Tokyo, Japan).

The status of necroptosis, pyroptosis, and autophagy was evaluated using an immunohistochemistry staining assay, as previously reported [50 (link)]. Paraffin sections of liver tissues were incubated with the primary antibody of p-MLKL (Abcam) [51 (link)], NLRP3 (Abcam) [7 (link)], or LC3 (Cell Signaling, Danvers, USA) [52 (link)]. All sections were scanned (TissueGnostics Axio Observer Z1 microscope; TissueGnostics GmbH, Vienna, Austria) and analyzed (Image J).

Paraffin-embedded sections (3 µm) were deparaffinized in xylene, rehydrated through ethanol washes and rinsed in PBS. Antigen retrieval was performed by pressure cooking for 6 min in citrate buffer, pH 6.0 (target retrieval solution, Dako, Santa Clara, CA, USA) followed by a blocking step using 2.5% BSA. Sections were stained overnight at 4 °C with the following primary antibodies: rabbit anti-α-SMA, rat anti-CD31 (both Abcam, Cambridge, UK). After three washing steps, secondary antibodies conjugated with DyLight Fluor 488 and DyLight Fluor 550 (Abcam, Cambridge, UK) were applied for 1 h at room temperature; 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma Aldrich, Vienna, Austria) was used for nuclear staining and slides were embedded in Permafluor mounting medium (ThermoFisher, Vienna, Austria). Images were taken on a Zeiss Axio observer Z1 microscope using TissueFAXS software (version 6.06.245.103, TissueGnostics, Vienna, Austria).
All histologic examinations were evaluated by an independent observer blinded for the subjects’ status and time point.

RAW 264.7 cells were seeded into Osteo Assay surface plates (Corning Inc., Corning, NY, United States) (Kim et al., 2019 (link)). The differentiation procedure was the same as mentioned above. On the 4^th day, the conditioned medium was removed, and the cells were treated with 5% sodium hypochlorite for 5 min. The plates were washed thrice with distilled water, and 5% (w/v) aqueous silver nitrate was added to each well for 30 min in the dark. The plates were removed from the silver nitrate solution and soaked in distilled water for 5 min. The water was discarded, and 5% sodium carbonate was added for 4 min. The sodium carbonate solution was removed, and the plates were dried at 50 °C for 1 h. Five random regions of each well were photographed using a TissueGnostics Axio Observer Z1 microscope (TissueGnostics GmbH, Vienna, Austria), and the resorption area of the chosen regions was counted using HistoQuest (TissueGnostics). $\mathit{P}\mathit{i}\mathit{t}\mathit{a}\mathit{r}\mathit{e}\mathit{a}\left(\%\right)=\mathit{p}\mathit{i}\mathit{t}\mathit{a}\mathit{r}\mathit{e}\mathit{a}/\mathit{w}\mathit{e}\mathit{l}\mathit{l}\mathit{a}\mathit{r}\mathit{e}\mathit{a}\times \mathbf{100}.$

The immunohistochemical assay, as per our previous reports [22 (link),23 (link)], was conducted to analyze the following crucial mechanisms, including inflammation, oxidation, and cell death processes of necroptosis and pyroptosis. In brief, lung tissue paraffin sections were prepared and then conjugated with one of the following primary antibodies (all from Abcam), including anti-iNOS (a marker for M1 phase macrophage polarization) [30 (link)], anti-CD206 (a marker for M2 phase macrophage polarization) [37 (link)], anti-MDA (a marker for lipid peroxidation) [38 (link)], anti-pMLKL (necroptosis-related protein) [39 (link)], and anti-NLRP3 (pyroptosis-related protein) [15 (link)]. All sections were observed (TissueGnostics Axio Observer Z1 microscope; TissueGnostics GmbH, Vienna, Austria) and scanned. The scanned images were then analyzed with the image processing software Image J.

The formalin-fixed lung tissue samples were processed, as above-mentioned. The tissue sections were then incubated with the primary antibody against macrophage activation (i.e., M1 phase polarization) related protein inducible nitric oxide synthase (iNOS) (anti-iNOS antibody, ab3523; Abcam), NLR family pyrin domain containing 3 (NLRP3) inflammasomes (anti-NLRP3 antibody, ab263899; Abcam), or malondialdehyde (marker for lipid peroxidation) (anti-malondialdehyde antibody, ab27642, Abcam) [27 (link)]. All sections were observed (TissueGnostics Axio Observer Z1 microscope; TissueGnostics, Vienna, Austria) and analyzed (Image J).