Signalstainr dab substrate kit

Paraffin-embedded sections of human and murine samples were cut into 4 µm sections with a microtome (Leica). Slides were de-paraffinized and rehydrated using the following protocol: 3 × 5 min in Xylene, 2 × 2 min in ethanol (100%), 2 × 2 min in ethanol (95%), 2 × 2 min in ethanol (70%), 2 min in ethanol (50%) and 5 min in H₂O. Antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 800 W, 650 W and 360 W for 5 min each. Samples were permeabilised with TBS 0.1 % Triton X-100 for 10 min and blocked with TBS containing 3 % H₂O₂ for 10 min. Human samples were stained with anti-SREBF2 (R&D, MAB7119), anti-USP28 (Sigma, HPA006778), anti-SREBF1 (PTG, 14088-1-AP) and anti-HMGCS1 (Abcam, ab155787). Murine samples were stained with anti-USP28 (Sigma, HPA006778), Nkx2-1 (TTF1; Santa Cruz sc-13040) anti-HMGCS1 (Abcam, ab155787), anti-SREBF2 (R&D, MAB7119), anti-Δnp63 (4A4) (Ventana, 05867061001) and anti-Cytokeratin 5 (Bimake, A5439). Slides were developed with DAB staining solution (SignalStainR DAB Substrate Kit, Cell Signaling 8059 S) and counterstained with haematoxylin (Sigma H3136). Slides were scanned at 40x resolution using a Pannoramic DESK II slide scanner (3D Histech) and analysed using QuPath (version 0.3.2).

After deparaffinage of paraffin sections in the 100% xylene and gradient rehydration (100%, 95%, 90%, 85% and 80% ethanol), the endogenous peroxidase activity was blocked by 3% hydrogen peroxide treatment for 10 min, and the sections were washed with phosphate-buffered saline (PBS) twice for 5 min each. The sections were then incubated with a MUC5AC antibody (dilution, 1:500; Abcam) in the primary antibody dilution buffer (Beyotime Biotech, China) for 12 h at 4°C. Following this, the sections were washed with PBS thrice for 3 min each, incubated with the IHC Detection Reagent (HRP, Mouse, Cell Signaling Technology) for 30 min at room temperature, and washed again with PBS thrice for 5 min each. The secondary antibody was detected with the SignalStain R DAB Substrate Kit (Cell Signaling Technology) after which the slides were counterstained with hematoxylin and mounted. To evaluate protein expression, semi-quantitative image analysis was conducted to measure the mean optical density (MOD) using the Image-Pro Plus software version 6.0.