Histocore pearl

Diethyl ether was used to anesthetize mice before post-fixation in Zamboni’s fixative (0.1 M phosphate buffer, 15% picric acid, and 2% PFA), which was then used to post-fixate the mice for H&E staining. The samples were then embedded in a tissue processor (HistoCore PEARL, Leica) for 24 h. The embedding process was 70% EtOH, 80% EtOH, 90% EtOH for 2 h each, 100% EtOH for 4 h, xylene for 8 h, and paraffin for 6 h finally embedded in paraffin. The retinas were longitudinally sectioned (7 µm) through the optic nerve. H&E staining was performed as previously described (Brahma et al., 2022 (link)). H&E-stained retinas were imaged under a DP71 microscope (Olympus). The RGCs were counted from the ON head to the Ora serrata. The number of cells in the ganglion cell layer (GCL) and the thickness of the IRL (from the internal limiting membrane to the inner boundary of outer nuclear layer) were measured (Sano et al., 2019 (link)). Moreover, IRL thickness was measured in the areas 0.5 mm and 1 mm from the ON head.

All samples were processed using Leica Histocore Pearl automatic processor. A total of 29 (SR N = 19, AF N = 10) and 18 patients (SR N = 13, AF = 5) were used to assess fibrosis and cardiomyocyte cross-sectional area, respectively. Paraffin inclusion was performed in Leica Histocore Arcardia Embedding System. Sections of 3 µm were cut and stained with hematoxylin–eosin (Harris hematoxylin and alcoholic eosin) for assessing cardiomyocyte cross-sectional area using Leitz Wetzlar-Dialux 20 microscopy and coupled camera (Olympus XC30). At least 60 cardiomyocytes were counted using the 250 × amplification; fibrosis was assessed using 3 µm atrial sections stained with Red Sirius and quantification was performed with Image-Pro Plus in 8 fields per sample using the 100 × amplification (results presented as percentage of the total area of each section).

The formalin fixed tumor samples were processed using the Leica HistoCORE PEARL automated tissue processor, following a 20-hour pre-programmed time-schedule. The tumor samples were wax embedded using the Leica EG1150H tissue embedder. The samples were sectioned into 3 μm sections and were used for hematoxylin and eosin staining (H&E) to assess tissue morphology. Images were acquired on an ImageXpress^® Pico (Molecular Devices) and CellReporterXpress^® software (Molecular devices) and whole-slide acquisition was performed using the 4X objective.

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).

Slices from the right hemisphere were processed through graded alcohols and xylenes for 20 h using the HistoCore PEARL (Leica, USA). Processed slices were embedded in paraffin wax using the HistoCore arcadia H (Leica, USA). Tissue blocks were cut in 6 µm serial sections using a Leica microtome and mounted on Menzel Gläser Superfrost Plus (Menzel, Germany) microscope slides.