Pertex mounting medium

Cryoprotected brain sections were washed in PBS, mounted on to gelatine-coated slides and dried overnight at room temperature. Sections were submerged in xylene, followed by decreasing concentrations of alcohol. Sections were rinsed in tap water and then incubated in haematoxylin for 5 min. Sections were washed in running tap water, dipped in acid alcohol (0.5% HCl in 70% ethanol) and then incubated in eosin for 5 min. Sections were washed in running tap water and then dehydrated in increasing concentrations of alcohol. Finally, sections were incubated in xylene for 5 min and coverslips applied using Pertex mounting medium (Cell Path Ltd, UK). Images of stained sections were captured using a Nikon N1 upright microscope (Nikon, Surrey, UK).

Immunohistochemistry was performed on a Leica Bond-III Autostainer, using the Bond Polymer Refine Detection System Kit (Leica Biosystem), according to the manufacturer’s instructions. Epitope retrieval was performed in ER2 buffer (EDTA based buffer and surfactant, pH = 9) for 30 min at 95°C for NRF2 staining and in ER1 buffer (EDTA based buffer and surfactant, pH = 6) for 20 min for NQO1 staining. Primary NRF2 antibody (Santa-Cruz Biotechnology A-10, sc-365949) was applied at 1:100 dilution for 1 h at room temperature. Primary NQO1 antibody (SIGMA-ALDRICH, HPA007308) was applied at 1:150 dilution for 20 min at room temperature. Slides were counterstained in hematoxylin and mounted in Pertex mounting medium (CellPath). Staining was categorized using a standard pathological system, with the staining intensity (+ to +++) and the percentage of tumor stained cells (0 to 100%). Samples were considered NRF2-high if nuclear staining intensity was +++ in more than 50% of tumor cells, and intermediate (+++/<50% and ++/>50%), low (++/<50% and +/>50%), or negative (+/<50% and 0).

Samples embedded in optimal cutting temperature media (OCT, CellPath) were cryo-sectioned at 10 μm. Gram-Twort, Acridine Orange (AO; Sigma-Aldrich), and Concanavalin A (ConA, Thermo Fisher Scientific) staining were used to visualize porcine wound biofilm load. A modified Gram-Twort stain was carried out. Here, sections were fixed in methanol at -20°C for 10 min, stained with Crystal Violet and Gram’s Iodine solutions (both Sigma-Aldrich), differentiated in 2% (v/v) acetic-alcohol and counterstained with a 0.2% (w/v) neutral red and 0.2% (w/v) fast green (9:1) solution (Sigma-Aldrich). Sections were differentiated again, rapidly dehydrated in 100% ethanol and mounted with Pertex^® mounting medium (CellPath). Images were captured at ×100 magnification on a Nikon E400 microscope with SPOT camera (SPOT imaging). For fluorescent visualization, methanol-fixed sections were incubated in AO solution (2 mg/ml) for 5 min at RT and rinsed in dH₂O, or incubated in ConA (50 μg/ml) at 4°C O/N and counterstained with DAPI (Thermo Fisher Scientific). Mowiol 4-88 (Sigma-Aldrich) containing DABCO (Thermo Fisher Scientific) was used for mounting. Fluorescent images were taken on a Zeiss Axio Vert. A1 microscope with AxioCam| cm1 camera (Carl Zeiss Microscopy Ltd) at ×40 magnification. Gram-Twort biofilm thickness analysis was performed in ImageJ v.1.6 (NIH).

5 μm FFPE-mouse mammary gland sections were immersed 3 × in xylene for 5 min, 3 × in absolute ethanol for 2 min, then rinsed in distilled water. After incubation in haematoxylin (Sigma) for 2 min, slides were washed under running tap water and dipped in Scott’s Tap water for 30 s. Slides were transferred to eosin (Dako, Eli, UK) for 2 min and rinsed thoroughly using running tap water. Stained tissue sections were de-hydrated through increasing concentrations of ethanol, immersed in fresh xylene for 3 min and finally mounted using Pertex mounting medium (Cell Path, Newtown, UK).

Slides with cytospins or pellet sections were removed from −20°C storage and allowed to reach room temperature. They were then flooded with 1% aqueous toluidine blue (BDH, UK) stain solution for 30 seconds and rinsed in tap water. Slides were left to air dry before mounting under glass coverslips with Pertex mounting medium (Cell Path Ltd).¹⁹

Adult zebrafish were sacrificed in accordance with the schedule 1 guidelines of of the Animal (Scientific Procedures) Act by excess anaesthesia then chemically preserved in 4% paraformaldehyde (PFA) in 1× phosphate buffered saline (PBS) for 2 days at 4°C. Fish were then washed with 1× PBS (3 × 3 minutes) and decalcified in 0.25 M EDTA (pH 8.0) for 2 days at room temperature. Following EDTA treatment, fish were washed with 1× PBS (3 × 3 minutes) then processed using Shandon Excelsior TM carousel-type tissue processor (Thermo Fisher Scientific). Paraffin-infiltrated tissues were carefully embedded in hot paraffin wax using a Tissue-Tek embedding station (Sakura) then allowed to set on a cold plate for 1 hour. Sections were cut at a thickness of 5 μm using RM2035 Microtome (Leica) and subsequently stained with H&E dyes using Thermo Scientific Shandon automatic slide stainer (Thermo Fisher Scientific). Following staining, sections were mounted using Pertex mounting medium (Cell Path) and left to dry overnight at room temperature. Images were acquired with a [20x/0.80 Plan Apo] objective using the 3DHistech Pannoramic 250 Flash II slide scanner and processed using 3DHistech Pannoramic viewer software.