Excelsior es tissue processor

Pituitary and organoids were fixed in 4% paraformaldehyde (PFA; Merck) and embedded in paraffin using the Excelsior ES Tissue Processor (Thermo Fisher Scientific). Sections were subjected to immunofluorescence staining as described earlier (Cox et al., 2019 (link)). Antigen retrieval in citrate buffer (Merck) was followed by permeabilization with Triton X-100 (Merck) and blocking with donkey serum (Merck). Following incubation with primary and secondary antibodies (Appendix 1—key resources table), sections were covered with ProLong Gold (Thermo Fisher Scientific) after nuclei counterstaining with Hoechst33342 (Merck).
To quantify SOX2⁺, Ki67⁺, PIT1⁺, and GH⁺ cells, dissociated AP cells were spun onto SuperFrost glass slides (Thermo Fisher Scientific) and the cytospin samples immunostained as described before (Fu et al., 2012 (link)). Proportions of immunoreactive cells were counted using Fiji software as previously described (Vennekens et al., 2021 (link)).
Images were recorded using a Leica DM5500 upright epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) accessible through the Imaging Core (VIB, KU Leuven) and converted to pictures for figures with Fiji imaging software.

Fixed tissues were processed in an Excelsior ES tissue processor (ThermoFisher Scientific, Runcorn, England, UK) for 4 h. After paraffin embedding in a cross-sectional orientation, 4 μm paraffin sections were cut and collected onto SuperFrost glass slides (ThermoFisher Scientific, MA, USA). Slides were baked for 2 h at 60 °C, deparaffinised in xylene, and rehydrated with decreasing concentrations of ethanol. Some of the serially sectioned slides were stained with haematoxylin and eosin to determine tissue orientation and structure; others were stained with PAS-Mayer’s haematoxylin to determine the distribution of goblet cells and to confirm the identity of the conjunctival tissue. The rest of the slides were stained with fluorochrome-labelled antibodies for immunofluorescence microscopy. Tissue samples excluded from immunofluorescent staining had folded regions, heavily rolled areas, inflamed epithelia defined by polymorph infiltration, missing goblet cells or large areas filled with erythrocytes from ruptured conjunctival capillaries.

Tissue samples of the skin and VCA transplants were fixed in 4% paraformaldehyde solution (PFA) for up to 24 hours, followed by processing for paraffin embedding using a Thermo Scientific^® Excelsior ES Tissue Processor. Sections of 4 µm thickness were prepared, deparaffinized with Xylene and a descending ethanol series, and stained with Mayer’s Hematoxylin and Eosin (both Morphisto GmbH, Frankfurt a.M., Germany, cat# 10231 and 11503) as described before (19 (link)). Sections were blinded and histopathological analysis was performed according to the respective Banff criteria for skin and VCA transplants (21 (link)).

All cranial explant samples were cut with an EXAKT saw to divide individual defects for analysis using paraffin and resin histology. One sample of each experimental group of both time points was decalcified with a rapid decalcifier (Kos Milestone microwave, ABACUS, Brisbane, Australia) at 37 °C. The demineralized samples were processed with an Excelsior ES Tissue Processor (Thermo Scientific, Scoresby, VIC, Australia) and embedded with paraffin wax at an embedding station (Thermo Scientific, Scoresby, VIC, Australia). Five-micron sections were obtained with a Leica RM2235 rotary microtome (Leica Biosystems, Nussloch, Germany).

After the dielectric measurement procedure was completed, seven samples of tissue used in MWA experiments in TMD Lab (1 cm to 2 cm wide and less than 5 mm thick) were taken from three lung samples (2 inflated, 1 not inflated) and embedded in formalin; three samples were taken from inside the ablation zone; three from not ablated tissue, and one included both ablated and not ablated tissue. For each sample it was noted the parent lung sample, if it was subject to inflation and ablation. Samples were embedded in formalin 2 to 4 h after ablation end.
The tissues were transferred into 4% paraformaldehyde (PFA) in PBS (pH.6.9) and stored at room temperature for at least 24 h until processing and embedding³¹ . The tissues were then processed in series of alcohols and embedded in Paraffin using Excelsior ES tissue processor (A82310100, Thermo Scientific, Waltham, MA, USA) and HistoCore Arcadia H embedding system (14039357258, Leica Biosystems, Wetziar, Germany).
The paraffin-embedded blocks were sectioned to obtain 5 µm sections and stained with haematoxylin and eosin (H&E), dehydrated, and mounted with dibutylphthalate polystyrene xylene (DPX). Images of the slides from H&E were acquired using Olympus Virtual Slide Scanner VS120-L100-W and OlyVia v3.3 (Olympus Life Science Waltham, MA, USA).