Avidin biotin peroxidase complex

Immunohistochemical staining of RKIP and phospho-Stat3 (Try705) in the cohort of clinical NPC tissues and xenograft metastases was performed on formalin-fixed and paraffin-embedded tissue sections. Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO). Finally, tissue sections were incubated with 3′, 3′-diaminobenzidine (Sigma) until a brown color developed and were counterstained with Harris' modified hematoxylin. In negative controls, primary antibodies were omitted.
The immunoreactions of RKIP and phospho-Stat3 were evaluated independently by two pathologists. Staining intensity was categorized: absent staining as 0, weak as 1, moderate as 2, and strong as 3. The percentage of stained cells was categorized as no staining = 0, <30% of stained cells = 1, 30~60% = 2, and >60% = 3. The staining score (ranging from 0-6) for each tissue was calculated by adding the area score and the intensity score. A combined staining score of ≤3 was considered to be low expression, and > 3 was considered to be high expression.

Expression of ICAM-1 and MCP-1 was assessed by immunohistochemical analyses as described previously [19 (link)]. Sections (thickness, 4 mm) were cut from paraffin blocks, mounted on polylysine-coated slides, and stained (H&E) for light microscopy. Sections were also dewaxed in xylene, rehydrated in a descending series of alcohols, and blocked for endogenous peroxidase and avidin/biotin activities. After blocking with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), sections were incubated with a rabbit polyclonal anti-mouse antibody against ICAM-1 and MCP-1 (1:150 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Sections were then washed and incubated with a secondary antibody (goat anti-rabbit) using the Evision + Labeled Polymer kit (Dako, Glostrup, Denmark) for 30 min followed by incubation with avidin-biotin–peroxidase complex (Dako) and development with diaminobenzidine chromogen for 5 min. Finally, sections were rinsed in distilled water, counterstained with hematoxylin (Dako), and mounted on glass slides before evaluation under the microscope. Semi-quantitative analyses of immunohistochemistry were undertaken by image analysis software (Image Pro Plus; Media Cybernetics, Rockville, MD, USA).

The kidneys were fixed in phosphate‐buffered formalin, embedded in paraffin and sectioned at 6 μm. Immunostaining was performed with an antibody against myoglobin (1:400, Dako, Carpinteria, CA, USA) and then with biotinylated secondary antibody (Dako) and avidin‐biotin‐peroxidase complex (Dako). The slides were counterstained lightly with haematoxylin. The immunoreactivity was screened semi‐quantitatively by randomly observing the number of myoglobin contained tubules in 15 high power fields of a whole section and estimating the proportion of myoglobin contained tubules of total renal tubules in each field.

Livers were collected from euthanized animals and submitted to fixation with 10% formalin. After paraffin embedding, 3-μm sections were obtained and submitted to hematoxylin–eosin (H&E) staining as directed by the manufacturer. Liver sections were immunostained with rabbit anti-insulin polyclonal antibodies (Cell Signaling, Danvers, MA) followed by incubation with the avidin–biotin peroxidase complex (DAKO, CA). Stained sections were imaged to observe pathological changes under a light microscope.