Citrosolv

De-waxed and hydrated paraffin sections were stained with haematoxylin for 8 minutes and then washed for 10 minutes in running tap water. Sections were stained in picro-sirius red for one hour. After two washes in acidified water, sections were dehydrated in three changes of 100% ethanol, cleared in Citrosolv (Fisher, Pittsburgh, PA), and coverslipped with toluene based mounting medium (Fisher, Pittsburgh, PA).

Slides were prepared according to the digital spatial profiling (DSP) FFPE Protein Manual (MAN-10100-05). FFPE slides were baked for one hour at 60 °C before being treated with CitroSolv (Fisher, 22-143-975) and descending concentrations of ethanol. Antigen retrieval in citrate buffer (pH 6) was performed in a pressure cooker and slides were then washed in TBS-T, blocked with Buffer W (Iba 2-1003-100) and incubated overnight at 4 °C with 62 UV-cleavable oligonucleotide-conjugated antibodies for DSP and fluorescent-labelled antibodies for visualisation (Pan Cadherin–Alexa Fluor 488, Syto83-Alexa Fluor 532, CD3e-Alexa Fluor 594, CD163-Alexa Fluor 647). The oligonucleotide-conjugated antibodies consisted of five panels (immune cell profiling, immune activation status, immune cell typing, tumour markers, and drug targets), and are shown in Table A4 of Appendix C.

Slides were de-paraffinized with Citrosolv (Fisher, Pittsburgh, PA), hydrated with graded ethanol, rinsed in tap water, immersed in 0.1% hematoxylin (Fisher) for 1–3 min, washed in tap water for 1 min, dehydrated through in graded ethanol (50%, 70%), immersed in 0.1% eosin (Fisher) for 1 min, and then dehydrated in 90% ethanol. Finally, sections were dehydrated in 100% ethanol and coverslipped with Permount (Fisher, Pittsburgh, PA).