Biotinylated anti rabbit secondary antibody

A human ovarian tissue microarray was obtained from Alena Biotechnology Co., Ltd. (Cat# OV1005a, Xi’an, Shanxi, China). All tissues were 10% formalin-fixed and paraffin-embedded. A total of 100 ovarian tissue specimens (20 normal controls and 80 ovarian tumors) were examined by immunohistochemistry (IHC). Among 100 specimens in a slide, seven came off during the IHC staining process. In the end, 20 normal controls (3 from normal ovaries and 17 from adjacent normal ovary tissues) with median age 48.5 years (range 19–63 years) and 73 ovarian tumors (12 benign, 7 borderline, 44 malignant, 10 metastatic) with median age 49.0 years (range 17–75 years) were statistically analyzed.
After blocking with 10% normal goat serum (Fuzhou Maixin Biotech Co., Ltd., Maixin Bio, Fuzhou, Fujian, China), the sections were incubated with rabbit monoclonal antibodies against STAT1 (Cat# 9175), pSTAT1-Y701 (Cat# 9167) and pSTAT1-S727 (Cat# 8826) (Cell Signaling Technology, Inc., Danvers, MA, USA), respectively, overnight, followed by incubation with biotinylated anti-rabbit secondary antibody (Cell Signaling Technology) at room temperature for 1 h. Scoring of STAT1 immunoreactive staining was performed by two independent examiners without any prior view of patient’s clinical data and classified as described previously using staining index (SI) system [21 (link)].

Immunohistochemistry was performed to study VEGF, MDR1, P-gp and p-TFAP2A proteins expression in Xenograft. In brief, paraffin-embedded specimens were cut into 4 μm sections and baked at 65°C for 30 min. The sections were de-paraffinized with xylenes, rehydrated, submerged into citrate buffer and microwaved for antigenic retrieval. They were then treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation with 3% BSA to block the nonspecific binding. Rabbit polyclonal antibody was incubated with the sections overnight at 4°C. For negative controls, the primary antibody was replaced by rabbit serum. After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody (Cell Signaling Technology, USA), and further incubated with streptavidin horseradish peroxidase complex (Cell Signaling Technology, USA). The degree of immunostaining of formalin-fixed and paraffin-embedded sections was reviewed and scored by two independent observers. The proportion of the stained cells and the extent of the staining were used as criteria of evaluation.

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)].