Histopathology and analysis of transgene expression were essentially as already described [5 (link)]. In brief, mouse mammary tissue specimens were fixed with 4% formaldehyde containing 1% acetic acid and embedded in paraffin. Deparaffinated sections were stained with hematoxylin and eosin. Immunostaining of SV40 large T-antigen was performed on paraffin sections using a triple-step immunoenzymatic method. Deparaffinated sections were reacted before antibody incubation with a commercial ‘target unmasking fluid’ (Dianova) in a microwave oven. Subsequently, sections were incubated overnight at 48°C with a 1 : 10,000 dilution of the polyclonal rabbit antiserum R15 against T-Ag [35 (link)]. Specifically bound primary antibody was detected using biotinylated anti-rabbit IgG and phosphatase-conjugated streptavidin from a commercial kit (Super Sensitive Detection System, Biogenex). Phosphatase enzyme activity was revealed with naphthol AS-BI phosphate in combination with hexazotized new fuchsine (Merck). Naïve rabbit serum served as control. Sections were slightly counterstained with hemalum. All photographs were taken by the Zeiss Axioplan2 imaging microscopic equipment with the camera ProgRes C12plus of Jenoptic using the Software ProgRes CapturePro 2.9.0.1.
Super sensitive detection system
Manufactured by BioGenex
Sourced in United States
The Super Sensitive Detection System is a laboratory equipment designed to detect and amplify target signals in various biological and chemical analyses. It provides a highly sensitive and specific detection method, enabling researchers to identify and quantify small amounts of target analytes with precision.
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Lab products found in correlation
2 protocols using super sensitive detection system
1
Transgene Expression in Mouse Mammary Tissue
2
Immunohistochemical Detection of p27 Protein
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Tissue sections were dewaxed in xylene and decreasing alcohols. Antigen retrieval was performed with 10 mM sodium citrate buffer at pH 6 in the microwave for 30 min. Endogenous peroxidase was quenched with 0.3% H 2 O 2 for 5 min. The sections were washed twice in TBS, incubated with blocking solution for 30 min and then with the primary antibody overnight at 4°C. The primary antibodies (Supplementary Table 1) were diluted in Dako REAL buffer (Dako). The anti-p27 antibody is raised against the full-length mouse protein and can recognize the mutated p27 protein in fibroblasts derived from MENX rats (Molatore et al. 2010a) . The supersensitive detection system (BioGenex, Freemont, CA, USA) was used and the immunoreactions developed in the DAB supplied with the kit. Washes between each step were done in TBS. Appropriate positive and negative controls were run in parallel to confirm the adequacy of the staining.
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