Superfrost plus slide

FFPE sections (5 µm) were transferred to superfrost plus slides (Thermo Fisher Scientific) and baked for 3 h at 60 °C. Before applying the in situ mutation detection method, the tissue sections were deparaffinized and pre-treated in 2 mg/ml pepsin in 0.1 M HCl (Sigma) for 30 min at 37 °C. The digestion was stopped with DEPC-treated H₂O (DEPC-H₂O) for 5 min followed by a wash in DEPC-PBS for 2 min. Thereafter, the tissue was dehydrated using an ethanol series of 70, 85, and 99.5% for 1 min each and stored at −80 °C until use for in situ mutation detection. One tissue section of the colorectal cancer samples was HE stained for histopathological confirmation of the diagnosis and representation of different tumor areas. The HE stained slide was used for orientation prior to mutation scoring by in situ mutation detection. For the tumor K255, only areas 1 till 3 were available for in situ mutation detection, as there was no tumor tissue left in deeper sections of the tumor block.
For cell line experiments, SW620 were seeded with media onto superfrost plus slides (Thermo Fisher Scientific) over-night at 37 °C with 5% CO₂. Cells were then washed with DEPC-PBS for 2 min, fixed in 4% m/V formaldehyde (Labonord, Templemars, France) for 15 min, washed with DEPC-PBS, and then dehydrated via a graded ethanol series and further processed as for tissue sections.

Brains were embedded in 5% wt. carboxymethyl cellulose (Millipore Sigma) in water and stored at −80 °C. For DESI, 20 µm thick sections were collected at −20 °C by using a CM1860 cryostat (Leica Biosystems). Superfrost Plus slides (Thermo Fisher Scientific) were used after cleaning with ethanol. For MALDI, 10 µm thick sections were collected on SiO₂ passivated, indium tin oxide coated polished float glass slides (Delta Technologies Limited, Loveland, CO, USA). Sections were dried under vacuum, stored at −80 °C until use, and thawed under vacuum immediately prior to analysis. Serial 50 µm thick sections were collected for extraction and LC/MS analysis after separating tumor-containing and non-tumor hemispheres (as described below).
Selected 10 µm thick tissue sections were mounted on Superfrost Plus slides (Thermo Fisher Scientific) in Fluoroshield mounting medium with DAPI (aqueous, Abcam, USA) and used for fluorescence microscopy to verify tumor location. A Leica DMi8 Thunder Imager was used to obtain images. For RFP, excitation was set to 540–580, DC was set to 585, emission was set to 592–668, and exposure time was set to 1.3 s. For DAPI, excitation was set to 375–435, DC was set to 455, emission was set to 450–490, and exposure time 59 ms.

10 mice (Charles Rivers, C57BL/6) were sacrificed following humane schedule one killing methods at 9 weeks old. 12 mouse eyes were fixed in Karnovsky's fixative for 3 h at 4 °C (Graham and Karnovsky, 1966 (link)). 8 mouse eyes were frozen on dry ice and cryosectioned transversely at 10 μm thickness using a Leica CM3050 S cryostat, collecting sections on Superfrost Plus Slides (Thermo Scientific, UK).
Eight human corneas containing the scleral ring were obtained from NHS Blood and Transplant (NHSBT). Cornea 1, from a 50 year old male, was dissected into quadrants and immersed in Karnovsky's fixative for 3 h at 4 °C and prepared for electron microscopy as below. Cornea 2 was from a 31-year old male, corneas 3 and 4 were from a 66-year old male, corneas 5 and 6 were from a 69 year old male, cornea 7 was from a 77 year old female and cornea 8 was from a 75 year old male. Cornea 2, 3, 4, 5, 6, 7 and 8 were dissected into quadrants, frozen on dry ice and cryosectioned transversely at 10 μm thickness using a Leica CM3050 S cryostat, collecting sections on Superfrost Plus Slides (Thermo Scientific, UK).