The Indiana University Animal Care and Use Committee approved the use of animals in this study and all procedures were performed as per NIH guidelines. Transformed cells with 50% basement membrane matrix (BME) type 3 (3632-005-02, Trevigen) (total 100 µL volume) were implanted into the mammary fat pad of 5–6-week-old female NSG (NOD/SCID/IL2Rgnull) mice. A 60-days slow release 17β-estradiol (0.72 mg) pellet (SE-121, Innovative Research of America) was implanted at the time of mammary fat pad injection. Tumor growth was measured weekly and tumor volume was calculated using the formula- sagittal dimension (mm) × [cross dimension (mm)]2/243 (link). The maximal tumor burden permitted by the Indiana University Animal Care and Use Committee is 2 cm × 2 cm. The maximal tumor burden was never exceeded during this study. After 2–4 months, tumors and lungs were collected and processed for hematoxylin and eosin (H&E), ERα, GATA3, FOXA1, EpCAM (MA5-29698, Invitrogen,1:500), CK5/6 (IR 780, Dako, Ready-to-use), CK8 (35bH11, N1560, Dako, 10 µg/ml), CK14 (LL002, ab7800, Abcam, 1 µg/ml), and CK19 (IR 615, Dako, Ready-to-use) staining43 (link).
Ma5 29698
Manufactured by Thermo Fisher Scientific
The MA5-29698 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for specific laboratory functions, but a detailed description cannot be provided while maintaining an unbiased and strictly factual approach. Additional information about the intended use or features of this product is not available.
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2 protocols using ma5 29698
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Xenograft Model of Breast Cancer
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Tumor Growth Dynamics and Marker Profiling
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Tumor cells (10,000, 50,000, 100,000, 1 × 106, or 5 × 106, total 100-μl volume) processed at 3% O2 and 21% O2 were implanted into the fifth inguinal mammary fat pad of each 6- to 8-week-old syngeneic FVB/N female mice without expanding in culture. Tumor growth was measured weekly, and tumor volume was calculated as described previously (55 (link)). Tumor tissues were harvested and divided into two fractions at their respective 3% O2 or ambient air. One fraction was collected for immunohistochemistry, and the other fraction was dissociated using a 10% collagenase/hyaluronidase enzyme mixture and processed at their respective 3% O2 or ambient air, and cells were either processed immediately for CSC marker profiles, reinjected into animals, or cultured at 5% O2 and 21% O2.
H&E, LGR5, TSPAN8, EpCAM, CK19, and CK14 immunostaining was performed at the CLIA (clinical laboratory improvement amendments)-certified Indiana University Health Pathology Laboratory, and the whole-slide digital imaging system of Aperio (ScanScope CS) was used for imaging according to a protocol described previously (55 (link)). The following antibodies were used: LGR5 (ab75732, Abcam), TSPAN8 (PA5-75311, Invitrogen), EpCAM (MA5-29698, Invitrogen), CK19 (IR 615, Dako), and CK14 (LL002, ab7800, Abcam).
H&E, LGR5, TSPAN8, EpCAM, CK19, and CK14 immunostaining was performed at the CLIA (clinical laboratory improvement amendments)-certified Indiana University Health Pathology Laboratory, and the whole-slide digital imaging system of Aperio (ScanScope CS) was used for imaging according to a protocol described previously (55 (link)). The following antibodies were used: LGR5 (ab75732, Abcam), TSPAN8 (PA5-75311, Invitrogen), EpCAM (MA5-29698, Invitrogen), CK19 (IR 615, Dako), and CK14 (LL002, ab7800, Abcam).
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