Sp rabbit mouse hrp kit

Paraffin-embedded sections were prepared, mounted onto slides, deparaffinized in xylene, rehydrated in a graded alcohol series and washed in deionized water. After antigen retrieval (sections were heated at 95-100°C on a hotplate for 30 min in 10 mM sodium citrate, pH 6.0), intrinsic peroxidase activity was blocked by incubation with 3% H₂O₂. Nonspecific antibody-binding sites were blocked using 3% BSA in PBS. Sections were then incubated with appropriately diluted primary antibodies specific for human or mouse CD31 (ab28364, Abcam), OSX (ab93876, Abcam), FGF2 (36,769, SAB), ATF6 (32,008, SAB) or GRP78 (3177S, CST) at 4°C overnight. Then, we followed the instructions of the SP Rabbit & Mouse HRP Kit (CWBio) for IHC. Alternatively, we incubated the slides with a fluorescence-labeled secondary antibody (CST). Slides were observed under a light microscope or a fluorescence microscope.

Paraffin-embedded sections were used for Survivin and ZIC1 immunohistochemical staining with a SP Rabbit & Mouse HRP Kit (CWBIO, Beijing, China). Survivin (bs-0615R) and ZIC1 (bs-11609R) rabbit polyclonal antibodies (BIOSS, Beijing, China) were both diluted at a concentration of 1:200 in phosphate-buffered saline (PBS). PBS without primary antibodies was used as negative control. Two pathologists (Qi-Feng Shi and Ye Zhang) independently evaluated the scores through a semi-quantitative assessment system and assigned the immunoreactivity score (IRS), which was obtained by combining the score of staining intensity (0, no staining; 1, mild staining; 2, moderate staining; and 3, strong staining) and the percentage of cells (‘0–100%’ = ‘0–10’). Any disagreement was resolved by discussion. The IRS was calculated by multiplying the staining intensity and percentage of cells, and the protein expression was considered positive only when IRS > 10.

A total of 241 breast tumors and corresponding normal tissues were collected. Three representative regions of each case were selected to obtain tissue cylinders with a diameter of 0.6 mm; we then arrayed these cylinders into a recipient block using a tissue chip microarrayer. Subsequently, we cut the recipient block into 5-µm sections on pretreated slides to support sample adhesion.
Rabbit anti-human ZIC1, ZIC2, ZIC3, ZIC4, and ZIC5 polyclonal antibodies (Bioss, Beijing, China) were used as the primary antibodies diluted at 1:100 in phosphate-buffered saline. A SP Rabbit & Mouse HRP Kit (CWBIO, Beijing, China) was used for the IHC. The slides were deparaffinized, rehydrated, and then boiled in a citrate buffer solution at a concentration of 10 mmol/L. After the solution cooled to room temperature, tissue chips were treated with blocking buffers and then incubated with the primary antibodies for 12 hours each. In sequence, the slides were marked by streptavidin with horseradish peroxidase (HRP), developed by diaminobenzidine, and counterstained with hematoxylin. Finally, we dehydrated and mounted these chips for storage and evaluation of the staining results.