Cryotome cm1100

Female 5- to 7-week-old NOD-SCID Gamma (NSG) mice were used according to protocols approved by the Johns Hopkins University Animal Care and Use Committee. Mice were anesthetized by the intraperitoneal (i.p.) injection of 100 mg/kg Ketamine, 16 mg/kg Xylazine, Vet One. MDA-MB-231 hypoxia fate mapping cells (2 × 10⁶⁾ were injected into the mammary fat pad (MFP) closest to the second nipple. Mice were i.p. injected with 1.25 mg of pimonidazole in saline (12.5 mg/mL) (Hypoxyprobe-1) 1 hr prior to sacrificing. Tumors were excised at various time points, formalin fixed (Sigma-Aldrich) for 1 hr, saturated in 30% sucrose (Sigma-Aldrich) at 4°C overnight, embedded in OCT media (Fisher Scientific), frozen in liquid nitrogen, sectioned via a cryotome CM1100 (Leica), and mounted onto Superfrost Plus microscope slides (Fisher Scientific). Tumor tissue sections were stained with DAPI (4′,6-diamidino-2-phenylindole) (1:1000 for 15 min, RT) and mounted with anti-fade solution. To assess the entire cross section of the tumor, slides were imaged with an Olympus (UPLFLN 4×) objective using Cytation 5 microscope (BioTek Instruments). Multiple image tiles were linearly stitched with Gen5 Software (BioTek Instruments).

An average of 500 μl of blood was collected by cardiac puncture into an EDTA tube by using a 26-G syringe needle. Red blood cells were lysed by using cold ACK Lysing Buffer (Quality Biological). Cells were incubated with 5 ml of the lysis buffer on ice for 10 min, followed by cold centrifugation at 1700 g for 5 min. This step was repeated, and the remaining cells were washed and resuspended in FACS buffer.
To assess and quantify metastasis, lungs were inflated with an OCT:PBS solution and excised for both image and flow cytometry analysis. For image analysis, lungs were formalin-fixed, saturated in 30% sucrose, embedded in OCT media, flash frozen in liquid nitrogen, sectioned via a cryotome CM1100 (Leica), DAPI-stained, and mounted for imaging. Full lung-slide sections were imaged as described for the primary tumor. Image analysis was performed by using NIS-Elements software. Individual metastatic events were carefully annotated. To measure metastasis size, metastatic events were marked as ROIs by using the auto-detect tool available in NIS-Elements. For flow cytometry analysis, lungs were chopped and digested (collagenase 2 mg/mL (Sigma-Aldrich), BSA 2 mg/mL (Gemini Bioproducts)) for 1 h at 37 °C at 160 RPM. After passing through a 70-μm strainer, cells were washed with PBS and resuspended in FACS buffer.