Axio scan z1 system

Fresh colon tissues were fixed in 4% paraformaldehyde (P1110, Solarbio, CHN) and embedded in paraffin. The sections were incubated with primary antibodies overnight at 4°C, and then HRP-conjugated secondary antibodies for 30 min at 25℃. Antibodies used in this study are listed in Additional file 1: Table S1. Subsequent detection was performed by substrate detection using DAB (8059, CST, USA). Images were taken using a BX51 microscope (Olympus, JPN), and AxioScan Z1 system (Zeiss, GER).

For histological analyses, tissues were fixed in 10% buffered formalin (Sigma‐Aldrich) and embedded in paraffin. Immunohistochemical staining with anti‐PD‐L1 antibody (rabbit monoclonal antibody (E1L3N); Cell Signaling #13684) was performed on 2.5‐μm tissue sections. Immunohistochemistry was performed using an automated protocol developed for the Autostainer Link automated slide staining system (DAKO, Agilent, Santa Clara, CA, USA). All steps were performed on this staining platform using validated reagents, including deparaffinization, antigen retrieval (cell conditioning), and antibody incubation and detection. Corresponding stainings were acquired and digitalized using the AxioScan.Z1 system (Zeiss). Digitalized images were automatically analyzed with the axiovision version 4.6.2 software (Zeiss). The percentage of PD‐L1 positivity was considered as ratio of PD‐L1‐positive cells to total number of cells.

Formalin-fixed paraffin-embedded sections were dewaxed in xylene and rehydrated in graded alcohols prior to antigen retrieval in Tris-EDTA pH9. Slides were blocked and incubated with primary antibodies at 4 °C overnight (CD31 (JC/70A, Abcam), Ki67 (EPR3610, Abcam), CD8 (SP16, Invitrogen), Granzyme B (NCL-L-GRAN-B, Leica), PD-1 (AF1086, RnD Systems), CD4 (EPR6855, Abcam), FOXP3 (236A/E7, Abcam), SMA (ab5694, Abcam), CD68 (KP1, Invitrogen)). Samples were washed and incubated in fluorescently conjugated secondary antibodies; nuclei were counterstained with DAPI. Whole slides were scanned at ×40 magnification on the Zeiss Axio Scan Z1 system. Image analysis was performed using HALO Software (Indica Labs, analysis algorithms: HighPlex FL v3.1.0, Object Colocalization FL v1.0, Area Quantification FL v2.1.5).

Whole sections were imaged to reveal the overall organ structure and distribution of LDs. Bright-field images of adrenal sections were taken under a 20× objective with an Axio Scan Z1 system (Zeiss). Nucleus staining was excited at 330–375 nm and fluorescence detected at 430–470 nm. LD staining was excited at 453–485 nm and fluorescence detected at 507–546 nm. Cell skeleton staining was excited with 540–557 nm and fluorescence detected at 578–640 nm. Autofluorescent signals were excited at 330–375 nm, with emitted signals detected over a broad specter of 430–640 nm.

Brightfield images were acquired with a Zeiss Axio Scan.Z1 system (Zeiss, Thornwood, NY, USA) at 40× magnification. Images of details were taken with a BX40 light microscope, DP72 camera, and cellSens Standard v1.16 imaging software (Olympus, Waltham, MA, USA).

Sirius Red staining was carried out for collagen I/III fibre-containing connective tissue on paraffin-embedded sections using a Picrosirius Red stain kit (Abcam, ab150681). Before staining, sections were deparaffinized in Histoclear and graded ethanol washes as already described (IHC staining section), then hydrated in distilled water. Sections were then incubated with Picrosirius Red solution for 60 min at r.t. and then rinsed twice with 0.5% glacial acetic acid solution (in dH₂O). Excess water was then removed by shaking the slides and then rinsing in 100% ethanol. Sections were then dehydrated by two washes of 100% ethanol for 2 min each and two washes in Histoclear for 2 min each. Coverslips were mounted and slides were imaged on a Zeiss AxioScan Z.1 system. Staining was quantified by thresholding the collagen-stained area for detection of fibres (red) and measuring this area relative to the total tissue area.

Staining for lipids was carried out on liver tissue in OCT that was snap-frozen in liquid N₂ and cryosectioned (15 µm). Sections were equilibrated to r.t. for 10 min and then stained with 0.5% Oil Red O solution (wt/vol, in isopropanol; Sigma, O1391) for 5 min, rinsed in tap water and counterstained with Mayer’s haematoxylin for 30 s. Sections were then rinsed again in tap water for 30 min and coverslips mounted. Images were acquired on a Zeiss AxioScan Z.1 system and ImageJ quantification of the Oil Red stain area was carried out relative to the background tissue area.