E0432

Expression of PTPN23 was analyzed by RNAscope multiplex fluorescence assay (Advance cell diagnostics, ACD, a BioTechne brand) following the manufacturer’s instructions. RNAscope was used for PTPN23 as it provides a very accurate and quantitative assessment of gene expression levels. PTPN23 targets the availability of EGFR protein. Therefore, EGFR protein expression was analyzed using immunofluorescence to focus on the spatial distribution of this receptor. Sections were dewaxed 3 × 10 min in xylene and rehydrated in ethanol series (100%, 90%, 70%, 50%, and 30%) for a period of 2 min for each step. Antigen retrieval was performed in citric acid 0.01 M at 90 °C for 30 min or Tris EDTA at 90 °C for 30 min. Blocking was achieved in PBS with 0.025% Tween20, 1% BSA, and 10% serum. Primary antibody (anti-pEGFR) was applied with an overnight incubation at 4 °C. Secondary Goat anti-Rabbit biotin was used 1/800 (Dako, E0432) followed by Streptavidin–HRP (Abcam, ab64269). The color reaction was performed in TSA buffer (100 mM Borate buffer with 0.0003% hydrogen peroxidase) with Opal-570 1/300 (Akoya Bioscience, OP001003) for 10 min. Slides were mounted with fluoroshield with DAPI as a nuclei stain.

Sections were dewaxed 3 × 10 min in xylene and rehydrated in EtOH series (100%, 90%, 70%, 50%, and 30%) for a period of 2 min for each step. Antigen retrieval was performed in citric acid 0.01M at 90°C for 30 min. Blocking was achieved in PBS with 0.025% Tween20, 1% BSA, and 10% serum. After ON incubation at 4°C, the primary antibody was washed in PBT 0.025%, and a secondary antibody was incubated for 1 h at RT. After washing, the slides were mounted with fluoroshield. Primary antibodies were anti-acetylated tubulin 1/300 (Sigma-Aldrich, T7451), anti-Plunc1 1/200 (R&D systems, AF4274), and anti-K5 1/300 (Biolegend, PRB-160P), and secondaries were Goat anti-Mouse-568 1/500 (Invitrogen, A11004), Donkey anti-Sheep-488 1/500 (Invitrogen, A11015), and Donkey anti-Rabbit 647 1/500 (Invitrogen, A31573).
For the detection of PCNA 1/300 (Abcam, ab19166) and P63 1/400 (Abcam, ab124762), a secondary Goat anti-Rabbit biotin was used 1/800 (Dako, E0432) followed by Streptavidin-HRP (Abcam, ab64269). The color reaction was performed in TSA buffer (100 mM Borate buffer with 0.0003% hydrogen peroxidase) with Opal-570 1/300 (Akoya Bioscience, OP-001003) for 10min. DAPI was used to stain nuclei. For each genotype, N = 3.

With antigen–antibody interaction, the targeted protein was visualized and detected using diaminobenzidine (DAB) (Dako Corp, Carpentaria, CA). Briefly, 5-μm tissue sections were removed of paraffin by soaking in xylene and were hydrated in a graded series of alcohol. Then, tissue sections were incubated overnight with an antibody against LSD1 (1:400). On the following day, after being incubated with goat anti-rabbit immunoglobulin (E0432, Abcam), staining was performed using the DAB chromogenic agent. Negative control experiments were routinely performed. The percentage of positive cells was determined by counting 500 cells within five high-resolution fields. IHC staining was evaluated using the percentage, which links IHC staining intensity (SI) with the percentage of positive cells (PP). SI was described by reaction scores between 0 and 3 (0 = none, 1 = low, 2 = moderate and 3 = strong). Accordingly, the number of positive cell nuclei was counted and scored between 0 and 3 (0 = none, 1 = ≤ 25%–40%, 2 = 41%–60%, 3 ≥ 60%). All patients were classified into two groups according to the IHC score: low expression level (0–3) and high expression level.