Hrp conjugated dab substrate

Formalin-Fixed, Paraffin-Embedded (FFPE) tongue and jejunum samples were sectioned onto Superfrost slides (Thermo Fisher Scientific, MA, USA), followed by deparaffinization and rehydrated according to standard protocols. Slides were then prepared for IHC with anti-Myeloperoxidase (MPO) (ab65871, Abcam, Australia) rabbit polyclonal antibody using mouse and rabbit specific HRP/DAB IHC detection kit—micro-polymer (ab236466, Abcam, Australia) according to manufacturer's instructions. Briefly, rehydrated slides were treated with hydrogen peroxide (Abcam, Australia), which was followed by heat-induced epitope-retrieval (HIER) with 0.1 M citrate buffer for 15 min. The sections were then incubated with the protein-blocking reagent (Abcam, Australia) for 10 min and then treated with a specific antibody for MPO (Abcam, Australia; 1:1250; 4 °C for overnight). Post PBS washing, the sections were incubated with goat anti-rabbit IgG secondary antibody (Abcam, Australia) at room temperature for 15 min and developed using HRP-conjugated DAB substrate (Abcam, UK), followed by counter-staining with hematoxylin. Grading was performed based on positive stain counting on the scanned digital images of the slides using QuPath open-source digital software v. 0.2.01^{125 (link)}.

IHC was performed as mentioned earlier^{59 (link)}. Briefly, 5-µm thick sections of tumors were deparaffinised, hydrated, and treated with peroxide (Abcam, UK ab236466), which was followed by heat induced epitope retrieval in sodium isocitrate buffer (pH 8 for p62). For p62, antigen retrieval was carried at 120 °C for 6 min in a pressure cooker. The sections were then incubated with the protein blocking reagent (Abcam, UK) and treated with anti-p62 (Abcam, UK; 1:100; 4 °C for overnight). The sections were further treated with the secondary antibodies and developed using HRP-conjugated DAB substrate (Abcam, UK). Grading was done based on the intensity and the extent of positivity as scored by an experienced pathologist.