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2 protocols using biotinylated donkey anti rat

1

Melanoma Immunohistochemistry Profiling

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Formalin-fixed paraffin-embedded tumor sections (5 μm) were immunolabeled for S100B to identify melanoma cells, and for Ki67 to identify proliferating cells. Sections were dewaxed in xylene and heat-treated in Target Retrieval Solution (DAKO). Non-specific antibody binding was blocked by incubating sections with 3% (v/v) hydrogen peroxide, biotin and avidin block (DAKO) and 10% (v/v) normal goat serum (DAKO). Rabbit anti-S100B (DAKO; 1:4,000) and rat anti-Ki67 (DAKO; 1:40) antibodies were then applied and incubated overnight at 4°C. S100B labeling was revealed with anti-rabbit HRP (DAKO) and AEC peroxidase substrate (Vector Laboratories), while Ki67 labeling was revealed with biotinylated donkey anti-rat (Jackson Lab; 1:300) followed by alkaline phosphatase-conjugated streptavidin (Rockland Inc.; 1:2000) and Alkaline Phosphate Substrate Kit III (Vector Laboratories). To visualize immune cells within the tumor bed we use anti-S100B (DAKO), CD45-APC (Biolegend; 1:200) and F4/80-biotin (AbDserotec; 1:50) antibodies overnight at 4°C. Anti-rabbit-FITC (DAKO; 1:500) and streptavidin-PE (Invitrogen; 1:500) antibodies were used to reveal S100B and F4/80 labeling respectively.
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2

Quantitative Analysis of pERK in 4T1 Liver Metastases

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Following 21 days of 4T1 breast cancer cell injection, livers were collected, fixed in 4% paraformaldehyde, and embedded in paraffin blocks and 4-μm sections were made. Slides were deparaffinized, and antigen retrieval was done using citric acid pH 6. Blocking for unspecific binding was done with 20% normal horse serum (NHS) and 0.1% Triton in PBS. Rat anti-CD45 (Bio-Rad; #MCA1031G) and mouse anti-pERK 1:100 (Sigma; #M8159) were diluted in 2% NHS and 0.1% Triton and were incubated overnight. Slides were then incubated with biotinylated donkey anti-rat 1:100 (Jackson ImmunoResearch; #712-065-153) and HRP-conjugated goat anti-mouse 1:100 (PerkinElmer; #NEF822001EA) diluted 2% NHS for 1.5 hours. Slides were then incubated with 1:500 OPAL 690 (Akoya Biosciences; #FP1497001KT) and streptavidin Cy3 (016-160-084; Jackson ImmunoResearch). Slides were imaged with a Leica Mi8 microscope equipped with a motorized stage and a Leica DFC365 FX camera. Single ×20 magnification images were tiled to receive a full scan of the tumor section. The quantification in the liver sections stained with pERK was done by ImageJ.
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