Pannoramic scan 2

Paraffin sections were deparaffinized and antigen-retrieved using a pressure cooker. Sodium-citrate buffer (pH 6) or Tris-EDTA buffer (pH 9) were used as an antigen retrieval solution, depending on the type of the antigen. The Sections were incubated with BLOXALL (Vector Laboratories) at RT, blocked with 3% horse (or goat) serum in PBS or PBST (0.3% Triton X-100 in PBS), and then incubated at 4 °C overnight with primary antibodies. The samples were washed and incubated with biotinylated IgG anti-mouse or anti-rabbit secondary antibody (Vector Laboratories; 1:200 dilution) for 30 min at RT. VECTASTAIN ABC kit and DAB was used according to the manufacturer’s protocol (Vector Laboratories). Sections were counterstained with Harris hematoxylin (Papanicolaou solution 1a, Merck) and mounted with mounting medium (DAKO). For hematoxylin and eosin slides, sections were stained with hematoxylin and eosin (BBC biochemical) according to the manufacturer’s protocol. For histological assessment of collagen deposition, trichrome staining was performed using a Masson Trichrome Staining Kit (Sigma-Aldrich). Immunohistochemistry images were acquired using a Pannoramic SCAN II (3DHISTECH) and analyzed by CaseViewer 2.2 (3DHISTECH).

Fractionated series of ten µm thick, formalin-fixed embedded WT and KO tissue sections were used. Mid-organ slices were stained with Alcian blue and Periodic-acid Schiff reagents. After manual determination of the different kidney regions, the sections were fully digitized at 20× magnification using a digital slide scanner (Pannoramic Scan II, 3D HISTECH Ltd., Budapest, Hungary) and exported (CaseViewer, Version 2.3, 3D HISTECH Ldt., Budapest, Hungary) as images with a pixel size of 0.24 µm. The different areas and stripes of the kidney [cortex, outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla (ISOM) and inner medulla/papilla (IM/P)] as well as all glomeruli were highlighted manually using GIMP (Version 2.10.2, The GIMP team, http://www.gimp.org) (Supplementary Figure S2). The area of these compartments was measured and the number of glomeruli was automatically counted using Mathematica (Version 11.3, Wolfram Research Inc., Champaign, IL, United States). Ratios of specific layers and total kidney area and glomeruli per area of cortex and total kidney were calculated and compared between WT and KO (n = 18 sections from 11 mice per group, left and right kidney of mice). Sections with poor quality were excluded from further analysis (4 sections per group).

Formalin-fixed paraffin-embedded lung tissues at 0, 6, 10 h were sectioned every 50 μm for generating six 5 μm tissue slices per sample and stained with hematoxylin-eosin-saffron (HES). The slides were imaged with a slide scanner (Pannoramic SCAN II, v3.0.2, 3DHistech, Medipixel Ltd, Budapest, Hungary) and analyzed by an external pathologist and a veterinarian in a blinded fashion. Five randomly selected high power fields (7x10⁴ μm² area) from 6 slides per sample were observed and scored by quantification of airway and alveolar polymorphonuclear cells and interstitial edema according to a reference scoring [11 (link)].

The inguinal white adipose tissue (ingWAT), epididymis white adipose tissue (epiWAT), brown adipose tissue (BAT) and skeletal muscle were taken immediately after mouse sacrifice. One half of the tissues were fixed in 4% formaldehyde overnight. The other half of the tissues were frozen at −80 °C for molecular experiments. Tissues embedded in paraffin were cut into 5 μm sections. After they were dewaxed, ingWAT, epiWAT and BAT sections were stained with haematoxylin and eosin (H&E). The size of adipocytes was determined by measuring of cell diameters. Immunohistochemical staining was performed with MSTN antibody (AF788; R&D Systems; 1:100). The secondary antibody was HRP-conjugated rabbit anti-goat IgG (H + L) (SA00001-4, Proteintech;1:200). Livers were frozen in OCT embedding medium and then stained with Oil Red O. The sections were photographed by a whole slide imaging scanner (Pannoramic SCAN II, 3 D HISTECH).

The kidney tissues were fixed in 4% paraformaldehyde solution, and embedded in paraffin. Then, the sections of paraffin-embedded kidney tissues were stained with periodic-acid-Schiff (PAS) and hematoxylin and eosin (H&E). A Digital Slide Scanner (PANNORAMIC SCAN II, 3D HISTECH) was used to scan the stained slides. The inflammatory score and fungal burden of the kidneys were assessed as previously reported [38 (link)–40 (link)].
For immunohistochemistry (IHC) assay, the renal tissues sections were stained with the indicated primary antibodies. The stained sections were scanned by a Digital Slide Scanner. The ImageJ software was used to quantify the proportion of positive cells. The primary antibodies used were as follows: SLC7A11 (xCT) (Abcam, ab307601, 1:500), GPX4 (Abcam, ab125066, 1:100), Ly-6G (Abcam, ab238132, 1:2000), F4/80 (Cell Signaling Technology, 70076T, 1:250) and CD11c (Abcam, ab219799, 1:100).

Organs from sacrificed mice were fixed in 4% formaldehyde solution (Duksan, UN2209) at 4 °C overnight. Specimens of 4% formalin-fixed tissue were embedded in paraffin using an auto processor and subsequently sectioned at 9 μm. Hematoxylin and eosin (H&E) staining was performed according to the routine protocol, and slides were scanned using the Pannoramic Scan II (3DHistech). Histological examination was performed in a blind manner by requesting a histopathologist from the Center of Animal Care and Use of Lee Gil Ya Cancer and Diabetes Institute.

H&E stained slides were scanned using the Pannoramic Scan II (3D Histech) and pictures were exported using the Pannoramic Viewer Software. Immunofluorescence stainings were captured using an inverted fluorescence microscope (Cell Observer, Zeiss) (Figs. 2, 5) or Pannoramic Scan II (Suppl. Fig. 2). For the immunofluorescence staining of HNSCC whole-mount slices (200 μm), tile scans and z-stacks were recorded via a confocal microscope equipped with an Airyscan mode (CD7 Cell Discoverer with an LSM900/AiryScan module, Zeiss). Maximum projections were generated. All fluorescence pictures taken with a Zeiss microscope were processed using the Zen Blue software or ImageJ.