IHC analysis of p140Cap expression was performed on formalin-fixed paraffin-embedded tissue microarrays, prepared with tumour breast specimens, using a mouse monoclonal antibody anti-p140Cap (Supplementary Fig. 1 ), which was used at a dilution of 1:1,000 following an antigen retrieval procedure in EDTA pH 8.0. Immunocomplexes were visualized by the EnVision+ HRP Mouse (DAB+) kit, DAKO (K4007), and acquired with the Aperio ScanScope system (Leica Biosystems). Informed consent was obtained from all subjects. For the purpose of correlation with clinical and pathological parameters, tumours were classified based on the intensity of p140Cap staining as p140Cap-Low (IHC score <1) and p140Cap-High (IHC score ≥1).
Envision hrp mouse dab kit
Manufactured by Agilent Technologies
Sourced in Denmark
The EnVision+ HRP Mouse (DAB+) kit is a reagent kit used for immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded tissue sections. The kit utilizes a horseradish peroxidase (HRP) polymer to detect the primary antibody, and 3,3'-diaminobenzidine (DAB) as the chromogen to visualize the target antigen in the tissue sample.
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2 protocols using envision hrp mouse dab kit
1
Immunohistochemical Analysis of p140Cap
2
Histopathological Assessment of Organ Samples
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For histopathology, attention was paid to sample for each type of organ similar parts in all rats. The samples were fixed in a 4% phosphate-buffered formaldehyde solution (Sigma-Aldrich, 40% solution diluted 10 times), processed routinely, paraffin-embedded, and sectioned at 5-μm thickness by a microtome. Sections were then stained with haematoxylin-eosin staining. The samples were light microscopically (Olympus BX50) assessed. For the immunohistochemical staining of macrophages, the monoclonal antibody CD68 (MCA341R; AbD Serotec, Biorad, France) was applied on paraffin sections of liver, spleen and lungs at a dilution of 1:100. For visualization Envision/HRP mouse (DAB+) kit (K4007; DAKO, Glostrup, Denmark) was used for immunolabeling. This kit also blocks endogenous peroxidase. To demask the epitope we used citrate buffer pH 6 (S2369; DAKO, Glostrup, Denmark) in a pressure cooker. The slides were evaluated qualitatively and quantitatively by the pathologist (e.g. number and concentration of microgranulomas).
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