Immunohistochemical staining kit

This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and it was conducted with approval from the Ethics Committee of Zhejiang Cancer Hospital (No. IRB-2015-176). Written informed consent was obtained from the participants. The Primary LUAD tissue and corresponding normal lung tissue used in this study were provided by the Biobank of Zhejiang Cancer Hospital. The primary antibody was a rabbit anti-human ZC3H12D polyclonal antibody purchased from Proteintech [24991-1-AP]. The immunohistochemical staining kit was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. Paraffin-embedded tissue samples were sectioned and placed at 72 °C for 30 min, then dewaxed and hydrated by xylene and ethanol. Antigens were heat retrieved with citrate buffer. After three washes, the slides were placed in 3% hydrogen peroxide solution for 5–10 min and then washed with running water twice and PBS for 5 min. The primary antibodies were added (1:100 dilution) and incubated overnight at 4 °C. After washing with water twice and PBS for 5 min, the secondary antibodies were added and incubated for 20 min, followed by washing, DAB staining, counterstaining, and mounting.

Endometrial tissues were fixed in 10% neutral-buffered formalin for 24 h and then routinely processed and embedded in paraffin. Tissue sections were de-paraffinized, rehydrated, then subjected to antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were incubated overnight in primary antibody at 4 °C, followed with immunohistochemical staining kits (Zhongshan Golden Bridge). Control sections were run concurrently with the experimental sections using nonspecific rabbit IgG (#sc2027, Santa Cruz Biotechnology, Inc.). Digital images were captured using a Leica DM 2000 microscope and LAS Core software (Leica Microsystems Limited, Wetzlar, Germany). Quantitative analysis of the relative protein expression levels in the epithelial cells and stromal cells of endometrium samples were determined according to the integrated optical density (IOD) of the digital images (×400) using Image-Pro Plus System 6.0 (Media Cybernetics, Inc., Silver Spring, MD, USA) in a blinded fashion as described previously.³³ (link) Signal density data for the tissue areas were obtained from three randomly selected fields of view and subjected to statistical analysis.