Finesse me

The specimens were fixed in 10% neutral w/v phosphate buffer 0.05 M and decalcified in 2% nitric acid and afterward 4% formic acid. Afterward, they were dehydrated with serial ethanol, embedded in paraffin, and cut into 3-μm sections. (FINESSE ME+, Thermo Scientific). The sections were stained with toluidine blue, hematoxylin–eosin and Weigert–Van Gieson stains and viewed under a light microscope. They were blindly scored by two different investigators according to the International Cartilage Repair Society (ICRS) visual histological scale. Immunohistochemistry for collagen type II was performed in all groups, using the EnVision Flex kit and the Autostainer Link (DAKO).

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).

Tissue samples were fixed in formalin for 24 h, automatically processed (Citadel 2000 Tissue Processor, Thermo Fisher Scientific, Waltham, MA, USA), and embedded in paraffin (HistoStar Embedding Workstation, Thermo Fisher Scientific). Five consecutive sections of 4 µm thickness were obtained for each case using a microtome (FinesseMe+, Thermo Fisher Scientific). One section was stained with hematoxylin-eosin (Gemini AS Automated Slide Stainer, Thermo Fisher Scientific), and the following four sections were placed in positively charged glass slides and used for further immunohistochemical studies.