Paxgene tissue fix

Liver and kidneys were harvested, fixed in PAXgene Tissue FIX (Qiagen, Hilden, Germany) for 1 day at room temperature and kept at 4 °C in PAXgene Tissue STABILIZER (Qiagen) until they were embedded in paraffin for histological studies. Staining was carried out on 4 μm paraffin-embedded tissue sections subsequently deparaffinized in xylene and rehydrated in decreasing alcohol concentrations. Slides were incubated in Mayer’s hematoxylin for 6 min and subsequently in eosin for 3 min. Slides were mounted using Tissue-Tek Prisma (Sakura, Alphen aan den Rijn, The Netherlands). Digital scans of slides were acquired with Nanozoomer NDP (Hamamatsu Photonics, Shizuoka, Japan) and analysed by an experienced clinical pathologist.

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).

4-Nitroquinoline N-oxide (4NQO, Sigma Aldrich-N8141) was dissolved in propylene glycol as a stock solution (4 mg/ml) and further diluted in drinking water to a final concentration of 50 μg/ml. 6-week-old female C57BL/6 mice were exposed to 4NQO-laced water for 16 weeks (refreshed weekly) after which all animal cages were reverted to plain water^{55 (link)}. At week 20, mice were either treated with ACPP-MMAE and IR as indicated in figure legends. Since the entire tongue was exposed to 4NQO, the whole tongue was delineated and given 5 Gy × 2 using PXI SmART irradiator with 225 kVp X-ray tube. IR was delivered as 2 parallel opposed fields. All mice were given a full oral cavity examination biweekly, and any observed pathological changes were documented. On week 22, animals were euthanized, and tissues and oral pharynx were fixed in PAXgene tissue FIX (Qiagen) at room temperature for 12 h. Tissues were then transferred to 70% ethanol and paraffin embedded. Tongue sections were H&E stained and histopathologically assessed for the number and size of carcinoma lesions per tongue by a qualified Pathologist (A.A.M.) who was blinded manner to treatment of each slide.