Dab enhancer

Stainings were performed using the Bond Polymer Refine Detection system (Leica Biosystems, Newcastle-Upon-Tyne, UK) and provided materials on a Leica Bond Max and Autostainer XL apparatus for DMT1 (Novus Biologicals, Abingdon, UK, H00004891-M01, 1:2000), ZIP8 (Protein Tech, Manchester, UK, 20459-1-AP, 1:500), ZIP14 (Atlas Antibodies, Bromma, Sweden, HPA016508, 1:1000), L-ferritin (Abcam, Cambridge, UK, ab69090, 1:2000), H-ferritin (Abcam ab65080, 1:4000), ferroportin (Abcam ab85370, 1:300) and HO-1 (Abcam ab13243, 1:100). After antigen retrieval (Bond ER1 or ER2), sections were incubated with primary antibody for 15 min diluted in Bond Primary Antibody diluent or Antibody diluent with Background Reducing Components (Dako Agilent, Stockport, UK) and secondary antibody for 8 min. Signal visualization was performed with Polymer Refine for 8 min, DAB for 10 min and DAB enhancer (Leica Biosystems). Afterwards, nuclei were counterstained with haematoxylin and images were taken with a Leica DM 2000 microscope connected to a Leica Microsystem Ltd camera, NanoZoomer whole slide imager (Hamamatsu Photonics, Welwyn Garden City, UK) or a VisionTek digital microscope (Sakura Finetek, Alphen aan den Rijn, NL). Appropriate negative control stainings were included for all primary and secondary antibodies (Supplementary Figure 1 and 2, respectively).

Formalin-fixed paraffin-embedded (FFPE) RA synovial tissue sections were stained with haematoxylin and eosin (H&E). For IHC, sections were incubated with antibodies specific for B cells (CD20, [Dako L26]; 1:20), T cells (CD3 [Cell Marque MRQ-39]; 1:50), macrophages (CD68 (Leica KP1); 1:100) and endothelial cells (vWF (Dako A0082); 1:600) diluted in Primary Antibody Diluent BOND (Leica Biosystems), using automated IHC following heat-mediated epitope retrieval and diaminobenzidine chromogen (DAB; Leica) or Bond Polymer Refine Red (Leica Biosystems) detection. For p53β, IHC was done manually following antigen retrieval (heat-mediated Tris–EDTA, pH 9.0) using rabbit polyclonal antibody ‘79.3’ (1:150) in van Gogh diluent (Biocare Medical) overnight at 4 °C, detection by EnVision Dual Link (Dako) followed by DAB (Dako) with DAB enhancer (Leica Biosystems).

A custom probe to the unique region of ∆133TP53 and TP53β was made by Advanced Cell Diagnostics (Advanced Cell Diagnostics, Newark, CA, USA). The probe was designed to Δ133TP53β reference sequence DQ186651.1 with the probes between nucleotides +97 and +277 unique to Δ133TP53 isoforms, but excluding the upstream AluJb repeat^{47 (link)}. To increase the amount of sequence available for probe stability, nucleotides +847–1001 were also included. Probes to VEGFA were used on human (reference number 423161) and mouse cells (reference 436961, Advanced Cell Diagnostics, Newark, CA, USA).
Formalin fixed paraffin-embedded cell clots and tumors were cut into 5 µm sections. The RNAscope method used the manual assay 2.5 protocol with Protease Plus reagent for protein digestion and the 2.5HD reagent kit brown for detection of the probe according to the manufacturer’s instructions. The assay was optimized using paraffin-embedded cell clots containing MCF7 and TP53 null Saos-2 cells (Supplementary Fig. S2). Following addition of DAB, DAB enhancer was added (Leica Biosystems, Wetzlar Germany). Positive cells were identified using the Aperio Scancope CS digital pathology system and quantified using the Aperio RNA ISH Algorithm (Aperio, Vista, California, USA). Slides were evaluated by two blinded examiners.