Dolph Houben

120 CHAPTER 6 Laser Capture Microdissection Laser Capture Microdissection (LCM) was used to remove a small segment of newly formed bone from the area between the Calcein and Tetracycline double labels for subsequent PCR analysis. Bone cross-sections were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek USA Inc., Torrance, CA) at -20⁰C. Five µm sections were made on a Leica CM3050S cryotome (Leica Biosystems Inc, Buffalo Grove, IL), placed on a PEN-membrane slide (MembraneSlide 1.0, Carl Zeiss Microscopy GmbH, Gottingen, Germany) and stored at -80⁰C. For LCM, slides were dehydrated following the Carl Zeiss PALM-protocol for DNA handling. New bone formation was visualized by fluoroscopy at a 20X magnification. Areas of new bone formation were selected, and laser captured using the PALM MicroBeam system (PALM Microlaser Technologies AG, Bernried, Germany) with the PALM Robo 4.8 software (Carl Zeiss MicroImaging, Munich, Germany) (Fig. 2). Using a stable proteinase K as an extraction agent (Arcturus PicoPure DNA extraction kit, Arcturus Biosciences Inc.), microdissected tissue sections (total of 160,000µm 2 ) were catapulted into the cap of a clean 0.6ml the tube containing 35mL of the extraction agent. Tubes containing the tissue and extraction agent were centrifuged and incubated following the manufacturer's protocol. The extracted genomic DNA was stored at -20⁰C prior to PCR analysis. Figure 2: Fluorescence microscopy image at a 10X magnification of fresh frozen bone section after laser capture microdissection: (A) indicating the area dissected, which included newly formed bone. (B) indicating a similar area of new bone formation identified by fluorescence of Calcein and Tetracycline.