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After scanning with CBCT, specimens were immediately fixed in 4% formaldehyde and preserved for at least 48 h. Subsequently, they were decalcified in a mixture of formic acid, acetic acid, and hydrochloric acid. During decalcification, the superficial bone became soft and was removed. This process was repeated until a 3-mm-thick bone slice with the three GP points exposed on the surface was acquired (Fig. 1c). The whole decalcification process took 7–14 days. Finally, the specimens were sliced into 4-μm-thick sections and stained with hematoxylin–eosin. The GP points dissolved during the staining process, and blank circles served as markers of their original positions.
The stained sections were scanned using a digital slice scanning device (NanoZoomer S60, Japan), and the boundary of bone invasion was delineated using NDP. View 2.7 software (Hamamatsu, Japan). The distances between the GP points were measured for specimens embedded in paraffin and specimens stained with hematoxylin–eosin. For histopathological specimens, bone invasion was defined as replacement of bone by an advancing tumor front. The presence of tumor cells in the inferior alveolar nerve fibers indicated positive nerve invasion. A senior pathologist determined the diagnostic result and delineated the boundary of bone invasion with NDP. View 2.7 software (Hamamatsu, Japan).

A hematoxylin and eosin stain was performed to assess the general morphology of the glabrous skin of PIEZO1cKO and wild-type littermate controls. Glabrous skin of PIEZO1cKO and wild-type animals was dissected, and the tissue was fixed in 4% formaldehyde. The skin was processed, embedded in paraffin, sectioned into 4 μm sections and dried at RT until subsequent staining at the MCW Histology Core. Rehydrated sections were stained in hematoxylin for 3 min, washed in Richard-Allan Scientific Signature Series Clarifier 1,2 (for 45 sec, dipped for 30 sec in 0.1% ammonia water (bluing agent)), stained in eosin for 30 s, washed four times using 100% EtOH and lastly rinsed in Xylene. Slides were scanned using a Hamamatsu Nanozoomer HT slide scanner (Hamamatsu Photonics, K.K., Hamamatsu City, Japan) and images were assessed using NDP.View 2 software (Hamamatsu Photonics).

Tissues were embedded in Optimal Cutting Temperature compound, and frozen histological sections were cut at 30 µm, mounted on polyethylene naphthalate (PEN) slides and fixed in 70% ethanol for 5 min, followed by two washes with PBS for 1 min each. Slides were manually stained in hematoxylin and eosin (H&E) using a conventional staining protocol. A subset of samples (PD44594c–h and PD44589f) were fixed in PAXgene Tissue FIX (Qiagen) according to the manufacturer’s instructions. Fixed tissue samples were embedded in paraffin using a Tissue-Tek tissue-processing machine (Sakura). No formalin was used in the preparation, storage, fixation or processing of samples. Processed tissue blocks were embedded in paraffin wax, sectioned to 10-µm thickness and mounted onto PEN slides (Leica). Tissue slides were stained using a standard H&E protocol. Slides were temporarily coverslipped and scanned on a NanoZoomer S60 Slide Scanner (Hamamatsu); images were viewed using NDP.View2 software (Hamamatsu).