Diva decloaker

Tissues obtained from US Biomax (Derwood, MD) were formalin-fixed, paraffin embedded, and sectioned at 5 μm. The sections were then deparaffinized, rehydrated in water, and prepared by heat-induced antigen retrieval by Diva Decloaker (Biocare Medical Cat. No. DV2004MX), and Decloaking Chamber (Biocare Medical). Sections were stained with Bsg (1 mg/mL, 1:500) or Mouse IgG1κ isotype control antibody (Biolegend Cat. No. 401407, 1:500) by using PowerVision+ poly-HRP IHC detection systems (Leica Cat. No.PV6106).

Formalin fixed, paraffin embedded sections were deparaffinized in xylenes and rehydrated through a series of graded alcohols. Tissues were processed for antigen retrieval by boiling in Diva Decloaker (pH 6.2, Biocare Medical). They were blocked in 10% donkey serum for 1 h to prevent nonspecific binding. The sections were then incubated overnight at 4°C in primary antibody (anti-CCR2, Novus Biologicals, 1:100 and anti-CD68, Biorad, 1:100) or control IgG (anti-rabbit and anti-mouse IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 1 h at room temperature, and sections were washed in phosphate-buffered saline (PBS), mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Leica fluorescent microscope system. In addition, hematoxylin and eosin (H&E) stains were performed on serial sections to analyze morphology and severity of the AAA tissues. The infiltrated inflammatory cells were counted by a clinical pathologist at 3 randomly selected 20x region-of-interest of AAA and sham-control tissue slides (n=4/group). Pentachrome stain was also carried out to characterize the collagen content of AAA and sham-control tissues.

Triple immunostaining for lung tumors was developed on an automated system (Intelli PATH, Biocare Medical, Concord, CA) using a novel antibody combination and a multiplex detection technology. The antibody combination of p40 (pink color nuclear stains), TTF1 (brown color nuclear stains), and Napsin A (brown color cytoplasmic stains) was applied simultaneously. Subsequently, mouse-HRP and rabbit-AP polymer detection systems were used to achieve triple immunostaining. Slides were deparaffinized, and antigen retrieval was performed with cell conditioning solution, pH 6.2 (Diva Decloaker, Biocare Medical). Primary antibody cocktail incubation was done for 45 minutes followed by polymer detection (MACH2 Double stain 2), and the test was visualized using chromogenic DAB and Wrap Red. Slides were counterstained with hematoxylin and dehydrated for permanent mounting. Unless specified all reagents were supplied from Biocare Medical, Concord, CA.