Immunostaining of mouse liver, spleen and lung specimens was performed as previously described with slight modification [10 (link), 17 (link)]. Briefly, after fixation with 10% formaldehyde, samples were embedded in paraffin, cut into 3.5 mm sections, mounted on glass slides coated with silane, deparaffinized in xylene and rehydrated through a series of ethanol solutions. The avidin–biotin complex method was used. For antigen retrieval, the sections were autoclaved at 98°C for 45 minutes in diluted immunosaver® (1:200). Then, specimens were incubated with normal rabbit serum (Dako; diluted to 1:75), and thereafter with a monoclonal antibody for F4/80 (CI:A3-1, Novus, Littleton, CO) (1:100) for 15 minutes using intermittent microwave irradiation [18 (link), 19 (link)]. Sections were then incubated with biotin-labeled rabbit anti-mouse IgG serum (diluted to 1:300; Dako) and a streptavidin–biotin detection kit (Ultra Tech HRP kit®, Beckman Coulter, Brea CA) sequentially. Finally, sections were stained with diaminobenzidin solution.
Ultra tech hrp kit
Manufactured by Beckman Coulter
Sourced in France
The Ultra Tech HRP kit® is a laboratory reagent used for the detection of target proteins in biological samples. It employs a horseradish peroxidase (HRP) enzyme-based detection system. The kit provides the necessary components to perform immunohistochemical or immunocytochemical analyses.
Automatically generated - may contain errors
2 protocols using ultra tech hrp kit
1
Immunostaining of Mouse Tissue Sections
2
Immunohistochemical Analysis of Engineered Cartilage
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The expression of COL II in the engineered cartilage was examined by immunohistochemical staining at 12 and 24 weeks after implantation. Briefly, paraffin sections were deparaffinised followed by hydration in ethanol solutions of decreasing concentrations (100% to 70%), and pre-treated with 0.1% of Proteinase K (Vivantis Technologies, Oceanside, California) at room temperature for 30 minutes. The sections were then treated with protein blocking agent ( UltraTech HRP Kit, Beckman Coulter France, Villepinte, France) for ten minutes and incubated with goat serum to block non-specific sites. The slides were washed in PBS three times and the sections were incubated at 37°C for one hour with mouse anti-collagen type II monoclonal antibody diluted in PBS (1:200), followed by incubation with 1:100 diluted horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Dako, Carpinteria, California) for 30 minutes. Colour was developed with diaminobenzidine tetrahydrochloride (DAB).
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