8 Laser capture microdissection for root study JX Sun et al to humans. Therefore, we used both mouse incisors and molars in this study to define the regulatory mechanisms associated with the root formation and determine whether the EMIs are critical for the development of root tissues.30–32 This information should provide valuable insights and thus inform periodontal tissue regenerative therapy and tooth (crown and root) regeneration in the future. MATERIALS AND METHODS Sample preparation As shown in Figure 1, lingual cervical loop (LCL) epithelial cells, adja- cent apical dental papilla (DP) and apical DF cells were collected from mandibular incisors of three CD-1 mice on P7. Odontoblasts (OD) and CB were collected from roots of mandibular first molars from three mice on P14 when molar roots are actively developing. The protocol for animal use was approved by the University of Washington Institutional Animal Care and Use Committee. All equipment used for surgical dis- section of mandibles was cleaned with RNaseZap (Applied Biosystem/ Ambion, Austin, TX, USA). After the erupted portion of mandibular incisors and excess bones were cut off (Figure 1a), the mandibles were embedded in an optimal cutting temperature (Tissue-Tek; Sakura Finetek USA, Torrance, CA, USA) block using standard cryomolds (Sakura Finetek USA) and stored at 280 6C until use. For P7 incisor cervical loop epithelial and adjacent mesenchymal samples, the frozen mandibles were sectioned sagittally at a thickness of 14 mm in a cryostat (CM3050 S; Leica Microsystems, Buffalo Grove, IL, USA) using a disposable tungsten carbide blade (TC-65; Leica Microsystems, Buffalo Grove, IL, USA). For P14 root OD and CB, the frozen mandibles were sectioned frontally at a thickness of 6 mm at 220 6C. Six sections were placed per polyethylene naphthalate membrane frame slide (MDS Analytical Technologies, Sunnyvale, CA, USA) and kept at 280 6C until staining and the laser capture microdissection were performed. Section, staining, laser capture microdissection and RNA isolation Cell nuclei were stained with cresyl violet. Briefly, sections on each polyethylene naphthalate membrane slide were fixed with 95% etha- nol for 1 min, stained with 2.8% cresyl violet (MP Biomedicals, Solon, OH, USA) in 75% ethanol, and dehydrated using the following series of alcohol and xylene: 95% ethanol for 1 min twice, 100% ethanol for 1 min twice and xylene for 5 min twice. The sections were Figure 1 Laser capture microdissection (LCM) of dental epithelia, adjacent dental mesenchyme, odontoblasts and cementoblasts from sagittal sections of mouse mandibular incisors and molars. (a) Schematic illustration of a mouse mandible (sagittal plane) during tooth development. Rectangular frame shows the incisor cervical loop and surrounding area used for LCM (b). Dotted vertical line indicates frontal section of the first molar for LCM (c). Solid vertical lines indicate the orientation and size of the embedded tissues used in this study. Note the excess bones out of this border was trimmed off to make the sectioning more efficient and with high quality. (b) Undifferentiated epithelial and mesenchymal cells associated with incisor root formation. Cell nuclei are stained with cresyl violet (purple) on sagittal sections of the 7-day postnatal mouse mandible. The following three cell populations were captured: LCL (epithelia, red dotted line outlined), DP (mesenchyme) and DF (mesenchyme). Left panel: 340; middle panel: 3200; right panel: 3200. (c) Terminally differentiated cells associated with molar root formation, including: OD (dentin forming cells, not captured yet) and CB (cementum forming cells). Left panel: 3100; right panel: 3200. Alv, alveolar bone; CEJ, cemento-enamel junction; CB, cementoblasts; DF, dental follicle; DP, dental papilla; LCL, lingual cervical loop; OD, odontoblasts. International Journal of Oral Science