Impress igg peroxidase kits

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 was inactivated by treating the specimens with 0.3% H₂O₂ in methanol at room temperature for 30 min. Then, the specimens were treated with 0.1% Triton X-100 for permeabilization. After treatment with a blocking reagent (Nacalai Tesque) at 4 °C for 30 min, the specimens were incubated with primary antibodies, anti-mouse mesothelin (295D, D053-3, MBL, dilution 1:100), at room temperature for 1 h or at 4 °C overnight. The sections were stained using ImPRESS IgG-peroxidase kits (Vector Labs) and a metal-enhanced DAB substrate kit (Life Technologies), according to the supplier's instructions. After counterstaining with haematoxylin, specimens were dehydrated and mounted. For actin staining, mesothelial cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, actin was stained with ActinGreen 488 Ready Probes Reagent (R37110, Molecular Probes). Then, after washing with PBS, the nuclei were counterstained with Hoechst 33342 dye (Dojindo). The stained cells were washed in PBS for observation.

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.

Harvested tumors were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Tumor sections were dewaxed with xylene and rehydrated with ethanol (100%–70%). Antigen retrieval was performed by boiling the specimens in Immunosaver (Nissin EM) diluted 1:200 for 45 minutes at 98°C. Endogenous peroxidase activity was quenched by incubation with 3% H₂O₂ for 30 minutes, and the sections were permeabilized with 0.1% Triton X-100 (MilliporeSigma) for 15 minutes. After blocking with Dako blocking reagent for 30 minutes, sections were incubated with primary antibodies overnight at 4°C in a humidified box. Sections were then incubated with secondary antibodies with ImPRESS IgG-peroxidase kits (Vector Labs) and DAB chromogen and counterstained with hematoxylin. Stained sections were imaged using a BZ-X700 microscope (Keyence).