Immunohistochemical Analysis of Phospho-p38γ

Immunohistochemical (IHC) staining was performed as described [18 (link)]. Briefly, tumor tissues and lungs were removed and fixed with 4% paraformaldehyde and then transferred to 30% sucrose. Tissues were sectioned at 10 μm thickness using a Cryostat Microtome (Thermo Scientific). Tumor tissues were stained with phosphorylated p38γ using Diaminobenzidine (DAB) kit. Briefly, tissues were incubated with 0.3% H₂O₂ in methanol for 30 min at room temperature and then treated with 0.1% TritonX-100 for 10 min. The sections were washed twice with PBS and then blocked with 1% BSA and 0.05% TritonX-100 for 1 hour. The sections were incubated with phospho-p38γ antibody (1:300) overnight at 4°C. Negative controls were performed by omitting the primary antibody. After being rinsed in PBS, sections were incubated with a biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for an hour at room temperature. The sections were washed three times with PBS and incubated with in an avidin-biotin-peroxidase complex (1:100) for an hour and developed in 0.05% 3,3′- Diaminobenzidine (Invitrogen) containing 0.003% H₂O₂ in PBS. To examine the lung metastasis, the sections of lung tissues were stained with eosin and images were recorded using an Olympus BX51 microscope.