Neutral buffered formalin

Tumors were harvested at the experimental endpoints, fixed in 10% neutral buffered formalin (Richard-Allan Scientific), embedded in paraffin, and cut into 5 μm sections. Slides were heated for 30 min at 60°C, deparaffinized, rehydrated with an alcohol series and incubated in Tris-EDTA antigen retrieval buffer (#E1161, Sigma-Aldrich) for 20 min in a pressure cooker on 95°C . Sections were washed in PBS, permeabilized in 0.3% PBS-Triton X-100 for 40 minutes, then blocked in PBS/0.1% Triton X-100 containing 2% BSA and 5% donkey serum (#D9663, Sigma-Aldrich). Primary antibodies were incubated overnight at 4°C in blocking buffer. The following primary antibodies were used: RFP (#6g6, ChromTech, 1:500), GFP (#ab5450, Abcam, 1:500), vimentin (#ab92547, Abcam, 1:500), cytokeratin 17 (#4543, Cell Signaling, 1:250), galectin-4 (#PA5-34913, Thermo Scientific, 1:500). After washing with PBS, tissues were incubated with secondary antibodies (#A-11055, #A-10042, #A-31571, ThermoFisher, 1:500) for 1 h at room temperature. After staining slides were counterstained with DAPI (#D9542, Sigma Aldrich, 5 μg/ml) for 10 min and cover slipped with Mowiol mounting reagent. Hematoxylin and eosin (H&E) were performed using standard protocols. Images were acquired using 20x or 40x objective on a Zeiss Axio Imager Z2 and ZEN 2.3 software.

An ETM study on C57BL/6 male mice (N = 5) at six weeks of age was conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee of UCSF, protocol number AN148450–01B using Waldo method and as described previously^{7 (link)}. After 14 days, the hemi-maxillae were fixed overnight in 10% neutral buffered formalin (Richard-Allan Scientific, Kalamazoo, MI) and stored in 70% ethanol aqueous solution at 4°C.

A first molar (M1) and the adjoining PDL and alveolar bone was sectioned from the left hemi-maxilla of an 8-week C57BL/6 mouse, and fixed overnight in 10% neutral buffered formalin (Richard-Allan Scientific, Kalamazoo, MI). The specimen was then washed three times for 20 minutes each in 1X PBS, and stored in 70% ethanol aqueous solution at 4°C. To perfuse the specimen with OsO4, it was placed in 4 ml of 1% OsO4 for 3 hours, then rinsed 5 times for 15 minutes each in 0.1 M sodium cacodylate buffer and transferred to 50% ethanol. The osmicated specimen was then embedded and ultrasectioned to 90 nm of thickness placed on a formvar/carbon-coated Ni grid (Electron Microscopy Sciences) grid and was visualized using STEM (Sigma 500 aSTEM detector, Carl Zeiss Microscopy, Pleasanton, CA).