Impact of the freezing system on morphology of fresh mammary tissue sections and on quality of RNA extracted. Alveolar (acini) structures lined by MECs (yellow arrows) can be easily distinguished after staining mammary tissue sections with Cresyl violet AMBION. Immediately after collection, mammary tissue samples were washed in cold PBS solution, cut in cube of 3-4 mm thickness, and frozen in four different conditions: pieces of mammary tissue were either directly introduced into 1.5-ml eppendorf tubes and frozen in liquid nitrogen (A), or in cold isopentane (-80°C) using the SnapFrost™ system (C), or embedded in OCT® contained in cryomold before to be immediately immerged in liquid nitrogen (B) or in cold isopentane (D). RNA quality (RIN) which was estimated by RNA 6000 Pico LabChip kit and Agilent 2100 Bioanalyzer, was identical (ranging between 8.5 and 9.5) whatever the freezing procedure, as illustrated in the electrophoreris profiles. Some large blisters (red arrows on Figure A and B) appear however in biopsy flash frozen in liquid nitrogen, mainly without cryoprotector, and morphological details are better seen on tissues frozen using the SnapFrost™ system (Magnification: ×60). The green arrow indicates the thermoplastic film stuck on epithelial cells to be captured.
Bevilacqua et al. BMC Cell Biology 2010 11:95 doi:10.1186/1471-2121-11-95