Pannoramic midi 2 scanner

Liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm. Microscopic analysis of whole slides was performed using a Pannoramic MIDI-II Scanner (3D Histech, Budapest, Hungary). Trichrome staining (Polysciences, Inc., Warrington, PA, USA, #25088-1) was scored by the authors according to Kleiner’s NAS system and by a deep-learning module established by Heinemann et al. [19 (link),20 (link)] High magnification tiles (299 × 299 px² at 0.44 μm/px) were obtained to analyze ballooning degeneration, inflammation and steatosis, and low magnification tiles (299 × 299 px² at 1.32 μm/px) were generated to analyze fibrosis.

The degree of immunohistochemical expression of the examined antigens was assessed using a digital method (QuantCenter software; 3DHistech). For this purpose, each whole slide was scanned using the Pannoramic MIDI II scanner (3DHistech) at 40 × magnification, and the resulting images were pre-prepared for further evaluation with CaseViewer (3DHistech). To determine the percentage of positive immune cells displaying membrane expression of CD45/CD3/CD4/CD8, CellQuant (3DHistech) software module was used. Representative images with the positive immunohistochemical reactions against selected antigens are presented in Fig. 2A.Figure 2

(A) Representative photos showing the distribution of CD3⁺, CD4⁺, CD8⁺ and CD45⁺ cells in normal and post-mortem intestinal mucosa. Particular attention should be drawn to clusters of lymphoid tissue, which seem to be the main source of immune cells infiltrating the lamina propria. Scale bars = 50 μm. (B−E) Comparison of immune cell numbers between normal colonic mucosa (HC; n = 16) and post-mortem samples (PM; n = 16).

Following coverslipping, brain sections were digitally processed using the Nikon Eclipse Ti microscopy system (Nikon Europe B.V., Amstelveen, The Netherlands). Twenty and forty× objectives were used for that, as well as three filters—FITC, DAPI and TRITC. Three sections that were stained with cresyl violet were digitized differently by being scanned with a Pannoramic MIDI II scanner (3DHISTECH Ltd., Budapest, Hungary) equipped with a 20× objective lens. Pannoramic Viewer 1.15.2 software was used to make snapshots of brain regions of interest (ROI) in the corpus striatum. To calculate immunohistochemical and histochemical staining data, we used an open-source scientific image processing software (ImageJ version 1.54f, Bethesda, MD, USA). In ImageJ, we selected an ROI of equal size for all analyzed samples. For densitometry, we calculated the mean intensity of staining and expressed it in arbitrary units. Six sections per animal were used for BrdU/DCX and GFAP quantification.

Slides were scanned using the PANNORAMIC MIDI II scanner (3DHISTECH, Budapest, Hungary) at 40× magnification. Subsequently, the digital slides were uploaded onto the digital platform DIPAP (DIgital PAthology Platform, Medica—Editoria e Diffusione Scientifica Srl, Milan, Italy) for remote consultation and expert board discussion.

To evaluate the transfer of human platelet-derived mitochondria to mouse BA-MSCs, BA-MSCs were seeded in 12-well glass slides with 2 × 10⁵ cells per well. The cells were cultured overnight until all cells adhered to the wall. MitoTracker Red CMXRos labeled mitochondria were cocultured with BA-MSCs. At 4 h and 8 h, the transfer of mitochondria into BA-MSCs was detected using Pannoramic MIDI II scanner (3DHISTECH, Hungary). Before detection, the cytoplasm of BA-MSCs was labeled with carboxyfluorescein diacetate and succinimidyl ester (CFSE) (#C1031, Beyotime), and the nucleus of BA-MSCs was labeled with DAPI (#C1002, Beyotime).

Tissue array slicing, dewaxing, rehydrating, antigen retrieval, and nonspecific binding blocking steps were performed as described above in the IHC staining assay. Next, the tissue sections were incubated with primary antibodies against CD8 (1:100, Abcam, ab178089) and LAG3 (1:1000, Abcam, ab209236) overnight at 4 °C, and then incubated with the corresponding secondary antibodies at room temperature for 50 min in the dark. Subsequently, the nucleus was stained with DAPI solution at room temperature for 10 min followed by intrinsic fluorescence quenching. Finally, the prepared section was scanned as high-resolution digital images using a Pannoramic MIDI II scanner (3DHISTECH Ltd., Budapest, Hungary). DAPI glows blue by UV excitation, CD8 was labeled with red fluorescence, and LAG3 was labeled with green fluorescence. The IRS of CD8 was calculated by multiplying the mean number of CD8 positive cells per high magnification by 0.6. The IRS of CD8 and LAG-3 double positive cells was calculated by multiplying the ratio of CD8 + LAG-3 + /CD8 + cells by 12.