Tomo adhesion microscope slides

Sectioning of frozen specimens was performed on a CM3050 S cryostat (Leica Biosystems, Nussloch, Germany) at a section thickness of 10 µm. The specimens were mounted with frozen milli-Q water on the sample holder. The chamber temperature was set to −20 °C whilst the sample was held at −16 °C. For each animal, all matched tissue sections of formalin-fixed and fresh frozen tissues were mounted adjacent on one slide to achieve highest comparability of the data. Formalin-fixed, paraffin embedded tissues were sectioned to a thickness of 10 µm at room temperature on a microtome (Finesse ME+, Thermo Scientific, Waltham, MA, USA) and straightened on a water bath held at 40 °C. The sections were fixed onto the slides by backing them for 1 h at 63 °C. Prior to all MSI experiments the paraffin was removed by washing the slides for 1 min in xylene [32 (link)] followed by immediate drying under nitrogen. Samples were prepared on non-conductive SuperFrost microscope slides (Thermo Scientific, Waltham, MA, USA) for DESI experiments and hematoxylin and eosin (H&E) staining, whilst samples prepared for MALDI experiments were mounted onto conductive ITO slides (Bruker Daltonik, Bremen, Germany). Tissue sections for lactate dehydrogenase staining were mounted onto TOMO Adhesion Microscope Slides (Matsunami Glass Ind. Ltd., Japan).

Gross inspection has been performed on embryos in order to assess any pathological change according to standardized post-mortem procedures^{46 ,47 (link)}.
Eggshell, embryo and yolk samples from all unhatched eggs both with macroscopic changes and from 3 specimens (1 embryo and 2 pipped eggs) without apparent macroscopic lesions were collected for additional analysis. The samples from eggshell, embryo and yolk for microscopic examination were fixed in 10% formalin, embedded in paraffin, sectioned at 5 µm and mounted onto TOMO Adhesion Microscope Slides (Matsunami Glass), stained with hematoxylin and eosin (HE) using a semi-automatic histo-stainer (Leica Autostainer XL, Leica Biosystems Nussloch GmbH). Additionally, periodic acid–Schiff (PAS) and Grocott’s methenamine silver stain were performed in case of suspected fungal infections. Additionally, standard bacteriological examinations were routinely performed on the above-mentioned fresh eggshell, embryo, and yolk samples.