Ultravision quanto detection system hrp kit

IHC was performed according to the staining protocol of the UltraVision Quanto Detection System HRP kit (Thermo Fisher Scientific, MA, USA). The CD68 primary antibody (Abcam, Cambridge, United Kingdom) was used followed by procedures of visualization. After counterstaining with hematoxylin, images were analyzed under a microscope.

The paraffin-embedded mouse liver tissue sections were deparaffined, rehydrated, and unmasked using the Trilogy reagent (Cell Marque, Rocklin, CA) with microwave heating for 10 minutes. After cooling to room temperature, all tissue sections were blocked with UltraVision Hydrogen Peroxide Block (Thermo Fisher Scientific, Waltham, MA) for 10 minutes at room temperature, followed by UltraVision Protein Block (Thermo Fisher Scientific) at room temperature for another 10 minutes. Anti-HBcAg antibody (b0586, 1: 800 dilution, DAKO, UK) was applied to the tissue sections for overnight incubation at 4°C before being thoroughly washed 3 times with 1× phosphate-buffered saline. The subsequent immunostaining was carried out using an UltraVision Quanto Detection System HRP kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

After animals’ perfusion, the primary tumor tissues were collected, kept in 3% paraformaldehyde and 3% sucrose solution, processed and paraffinized. Lung sections were stained for hematoxylin and eosin (H&E) to detect clear metastatic foci. Tissue sections were also stained for metastatic common markers such as E-cadherin (1:100—Bioss), N-cadherin (1:100—BD Biosciences), MMP-9 (1:100—Bioss), MMP-2 (1:200—Neomarkers), CD44 (1:100—Abcam) and CD24 (1:100—Santa Cruz). In brief, the sections were de-paraffinized, retrieved by boiling, incubated with protein block followed by primary antibody diluted in PBS, and kept at 4°C overnight. Then, the sections were washed with PBS and incubated with the secondary antibody and HRP Polymer Quanto (Ultravision Quanto Detection system HRP kit, Thermo Scientific) as recommended by the suppliers. Then, the sections were rinsed with PBS, incubated with diaminobenzidine tetrachloride (DAB) substrate, counterstained with hematoxylin, dehydrated, and coverslipped.
For analysis, five randomly selected areas were photographed at 40x magnification from each section and quantified the number of labeled and unlabeled cells per area determined by two independent observers using Image J software (NIH).