Protein block serum free blocking reagent

Lung tissue samples for immunohistochemistry were collected from C57BL/6J mice. Tissue samples were fixed with formalin and embedded in paraffin. Following dewaxing and rehydration, heat‐induced epitope retrieval was performed by boiling the specimens in 1/200 diluted ImmunoSaver (Nissin EM, Tokyo, Japan) at 98°C for 45 min. Endogenous peroxidase activity was inactivated by treating the specimens with 3% H₂O₂ at room temperature for 10 min. Specimens were then treated with 0.1% Triton X‐100 for tissue permeabilization. After treatment with a Protein Block Serum‐Free blocking reagent (DAKO, Code X0909) at room temperature for 30 min, the specimens were incubated with primary antibodies, anti‐rabbit β‐Catenin (CST, 8480), anti‐ ‐mouse p16 (abcam, ab54210), and anti‐rabbit p21 (CST, 2947) at room temperature for 1 h or at 4°C overnight. The sections were then processed using ImPRESS IgG‐peroxidase kits (Vector Labs) and a metal‐enhanced DAB substrate kit (Life Technologies), according to the supplier's instructions. Haematoxylin and eosin (HE) and Masson's trichrome staining were performed on paraffin embedded tissue sections. After counterstaining with haematoxylin, specimens were dehydrated and mounted.

Lung samples were initially fixed in formalin and embedded in paraffin. Following the dewaxing and rehydration process, heat-induced epitope retrieval was performed by immersing the specimens in 1/200 diluted ImmunoSaver solution (Nissin EM, Tokyo, Japan) at 98 °C for 45 min. Endogenous peroxidases were inactivated by treating the specimens with 3% H₂O₂ at room temperature for 10 min. The specimens were then permeabilized with 0.1% Triton X-100. After treatment with a protein block serum-free blocking reagent (DAKO, X0909) at room temperature for 30 min, the specimens were incubated with the primary antibody at room temperature for 1 h or at 4 °C overnight. The sections were stained using ImPRESS IgG-peroxidase kits (Vector Laboratories) and a metal-enhanced DAB substrate kit (Life Technologies), following the manufacturer’s instructions. After counterstaining with hematoxylin, the specimens were dehydrated and mounted.