Pannoramic digital slide scanner

Eosinophils in the small intestine were identified by Hematoxylin-Congo red staining as previously described (20 (link)). Briefly, sections were stained in Hematoxylin for 5 minutes at room temperature and followed by rinsing in running tap water. Then, 0.5% alcoholic Congo red solution (C6277, Sigma-Aldrich) was added for staining for 15 minutes at room temperature. Finally, 75% alcohol solution was used to differentiate the sections for a few seconds. Eosinophils were calculated by counting the orange-red cytoplasmic cells in sections of the small intestine with the assistance of PANNORAMIC Digital Slide Scanners and CaseViewer (3DHistech, Budapest, HUN) and expressed as eosinophils/mm². Four to five section areas were randomly selected per animal for eosinophil assessment. Tissue sections were also prepared for H&E staining to observe the tissue structure and inflammation.

Kidneys were fixed in 10% buffered formaldehyde, embedded in paraffin, and processed for sectioning. Pathological changes were detected by hematoxylin and eosin staining. Glomerular lesions and mesangial matrix expansion were evaluated with PAS (periodic acid/Schiff) staining to verify that the DN model in STZ-induced SD rats was successfully constructed. Image acquisition was performed using Pannoramic Digital Slide Scanners (3DHISTECH Ltd.). Prefixed with a 3% glutaraldehyde, then the tissue was postfixed in 1% osmium tetroxide, dehydrated in series acetone, infiltrated in Epox 812 for a longer, and embedded. The semithin sections were stained with methylene blue and the ultrathin sections were cut with diamond knife, and stained with uranyl acetate and lead citrate. Sections were examined for microscopic glomerular lesions with JEM-1400-FLASH transmission electron microscope.

Mouse brains were embedded and cut into 15‐μm sections, followed by deparaffinization. The sections were then rehydrated in a solution of LFB (0.01% LFB in 95% ethanol) and incubated overnight at 56°C. After washing in 95% and 75% ethanol and removing excess stain, the sections were differentiated in lithium carbonate solution for 15 s, followed by washing in distilled water. Subsequently, the sections were dehydrated and fixed. Images were scanned using PANNORAMIC Digital Slide Scanners (3DHISTECH Ltd, Hungary). Quantification of LFB staining was performed within the corpus callosum zone of three mice per group using the image analysis system Image‐Pro Plus 6.0 (IPP 6.0, Media Cybernetics, Bethesda, MD, USA). The LFB intensity was measured and normalized to the corresponding area to obtain the final calculated results.

Mice were anesthetized and transcardially perfused with Phosphate buffered saline (PBS) and then Bouin’s solution (Sigma-Aldrich). After post-fixing in Bouin’s solution, the brain tissues were paraffin embedded. The paraffin sections were applied for hematoxylin and eosin (H&E) staining. The stained sections were scanned with Pannoramic Digital Slide Scanners (3DHISTECH).

Freshly collected tumour tissues were placed in 10% NBF and fixed for 24 h at RT followed by trimming to the thickness which did not exceed 3–5 mm. After rinsing with running water, the specimens were transferred to the vacuum tissue processor (HistoCore PEARL, Leica) for dehydration, then embedded into FFPE blocks using Tissue Embedding Center (EG1150, Leica). FFPE blocks were sectioned with a manual rotary microtome (RM2235, Leica), 4 µm thickness/section. Sections were processed for staining with hematoxylin and eosin (H&E), immunohistochemistry (IHC), or immunofluorescent (IF) analysis. For IHC, sections were stained with primary antibodies specific for anti‐PD‐L1 (ab174838F) and CD8 (#98941) or IF sections were stained with primary antibodies specific for CD31 (ab28364), NG2 (AB5320), and cell nuclei were counterstained with DAPI. All stained sections were scanned with Pannoramic Digital Slide Scanners for 40× magnification (3DHISTECH, Pannoramic SCAN). All the images were analyzed with HALO platform where tumour area and large areas of necrosis were quantified. Non‐tumour tissue on the periphery was excluded. Elongated blood vessels and pericyte co‐localization was quantified using Visiopharm platform. Quantitative histological analyses were performed at OracleBio Ltd. (Scotland, UK).