Histofine max po kit

Lung tissues were fixed with 10% formalin, embedded in paraffin and sectioned at a 3 μm thickness. Immunohistochemistry for Axl and Gas6 was performed using the Envision polymer method (Histofine MAX-PO kit, Nichirei, Tokyo, Japan). Heat-induced antigen retrieval by autoclave was performed using basic retrieval solution (R&D, cat# CTS013) for 5 min at 121 °C for the Axl and Gas6 staining. The sections were blocked with 10% rabbit serum for Axl and Gas6 staining and were then incubated at 4 °C overnight with the following primary antibodies: goat anti-human Axl antibody (R&D, cat# AF154, 1:100); goat anti-human Gas6 antibody (R&D, cat# AF885, 1:20). Endogenous peroxidase activity was blocked with 0.3% H 2 O 2 in methanol. The sections were subsequently reacted with a sufficient quantity of secondary antibodies conjugated with polymer and peroxidase (Nichirei). The immune complex was visualized with a 3,3'-diaminobenzidine (DAB) solution. Hematoxylin was used for counterstaining the nuclei. Nonreactive immunoglobulin of the same isotype was used as a negative control. Images were captured using a Nikon Eclipse microscope (Nikon). Information on background of patients for the histological analysis is shown in Table 2.