Superfrost charged slides

Formalin-fixed and paraffin-embedded (FFPE) archival tumour blocks were analysed by immunohistochemistry by collecting 4 μm sections on Superfrost charged slides (ThermoScientific). After drying overnight at 37 °C, samples were processed using a Ventana Benchmark immunohistochemistry platform (Roche) with antibodies against p53 (Agilent cat#7001, RRID: AB_2206626, 1:50), CK7 (Agilent cat#7018, RRID: AB_2134589, 1:250), PAX8 (Roche cat#760–4618, 1:100). Heat induced epitope retrieval was performed using CC1 (Roche), incubating samples at 95 °C for 36, 52, and 40 min for p53, CK7 and PAX8, respectively. Antibodies were incubated at 37 °C for 32, 40 and 32 min for p53, CK7 and PAX8, respectively. p53 and CK7 were detected using Ultraview universal DAB kit (Roche), while PAX8 was detected using Optiview universal DAB kit (Roche), all as per manufacturer’s instructions. Sections were counterstained using Haematoxylin II (Roche) for 12 min and bluing reagent (Roche) for 8 min. Slides were imaged using the EVOS FL Auto 2 Imaging System (Invitrogen), using a × 10 or × 40 objective lens under bright field, and processed using Adobe Photoshop.

The liver and kidney pro-inflammatory markers, TNF-α and IL-6, were measured using the commercially available ELISA kits according to the manufacturer’s instructions (OxiSelect Cell Biolabs, CA, USA) and the amount of total protein was determined by a BCA protein assay kit.
Moreover, sections on charged slides (Superfrost charged slides, Thermo Scientific, Braunschweig, Germany) from liver and kidney tissues were immunohistochemically stained with anti-NF-κB and anti-caspase-3 (Santa Cruz Biotechnology, CA, USA, respectively) using an Ultra Benchmark machine (Roche, Tucson, USA) and an Optiview Detection kit with haematoxlin as the counterstain. The percent of positively stained brown nuclei and cytoplasm for NF-κB and caspase-3, respectively, were examined in five microscopic fields (at x400 under Zeiss light microscopy, Jena, Germany).

After perfusion, processing in 30% sucrose, and embedding in Tissue-Plus OCT compound, whole embedded spines were stored at −80°C until sectioning. The entire spinal cord was sectioned at 50 μm on a cryostat (Leica CM1850) and then collected onto Superfrost charged slides (Fisher Scientific, Hampton, New Hampshire, USA, 12-550-15). Every 8th section was stained. The sections were thawed at room temperature for 10-min, prior to immunostaining. Sections were first blocked in 10% normal donkey in TNT buffer. The sections were then incubated for 48-h in primary antibodies against RFP and GFP (Supplemental Table 1). Following three rinses in TNT buffer, primary antibody staining was visualized using fluorescently-tagged secondary antibodies. The sections were rinsed again with TNT buffer and incubated in DAPI for 5 min before mounting with Fluoromount-G. The sections were imaged with a Hamamatsu 2.0-HT NanoZoomer at 20x magnification. Spinal cord sections were analyzed using the Allen Spinal Cord Atlas as a reference database (34 ).