Immunohistochemical Analysis of CALR Expression in NKTCL

We performed IHC analysis to determine CALR expression in the formalin-fixed paraffin-embedded human NKTCL and reactive lymphoid hyperplasia specimens. Briefly, the 4-μm thick specimens were deparaffinized with xylene, rehydrated, and incubated with 3% hydrogen peroxide to block endogenous peroxidase. Then, the sections were incubated overnight at 4° C with the monoclonal rabbit anti-CALR antibody (1:200 dilution; Cat. No. 12238; Cell Signaling Technology, Beverly, MA, USA). Then, the samples were incubated with the biotinylated anti-rabbit secondary antibody (1:150 dilution; Cat. No.14708; Cell Signaling Technology) for 1 h at room temperature followed by DAB staining (Cat. No. P0203; Beyotime, Shanghai, China). The samples were counterstained with hematoxylin and eosin (HE). The sections were examined under the inverted microscope (TE2000 –U, Nikon, Japan) and scored as previously described [44 (link)].