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Preparation of Thin Frozen Sections from Nonfixed and Undecalcified Hard Tissues Using Kawamoto’s Film Method (2020)

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Skeletal Development and Repair

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2230))

Abstract

A method for preparing frozen sections with an adhesive film is described. In order to observe fine structures and weak fluorescence of samples, new types of adhesive films [Cryofilm type 3C(16UF) and 4D(16UF)] are used. The adhesive film is made with very clear and very low autofluorescence. For gene analysis, a very thin adhesive film (LMD film) is used to cut by means of the laser microdissection (LMD). For MALDI mass spectrometry imaging (MALDI-MSI), a conductive adhesive film (Cryofilm type MS) is used to avoid electric charge of the sample. A biological sample is frozen quickly and freeze-embedded. The frozen sample is cut with a very sharp disposable blade made from fine tungsten carbide. The combination of the adhesive films and the blade can generate 3 micrometer thick sections from samples including bone, while it is also possible to generate 1 μm thick sections. The morphology of bone and soft tissues are preserved using this method. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly observed with an oil immersion lens at high magnification. Sections generated using the Cryofilm type 3C(16UF) shows weak fluorescent signals more clearly than sections generated with the previously reported adhesive films [Cryofilm type 2C(9) and 2C(10)]. Furthermore fluorescence of the fine structures in cells is clearly shown using a super–high-resolution microscope. Several staining and experimental methods such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization can be performed on these sections. This method is also useful for preparing frozen sections of large sample such as a whole-body mouse and rat. In gene analysis, gene quality of sample collected from the section made with the LMD film is superior to that of sample made by a conventional method. The Cryofilm type MS makes almost complete section from tissues including hard tissues and large samples. The satisfactory signals are detected from the section with MALDI-MSI.

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References

  1. Mullink H, Henzen-Logmans SC, Tadema TM et al (1985) Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies. J Histochem Cytochem 33:1103–1109

    Article  CAS  Google Scholar 

  2. Hosoya A, Hoshi K, Sahara N et al (2005) Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections. Histochem Cell Biol 123:639–646

    Article  CAS  Google Scholar 

  3. Nakano U, Kawamoto T, Takano Y (2001) Phosphatase activities of rat intestinal enterocytes and its relation to diverse luminal pH, with special references to possible localization of phytase along the brush border membrane. Arch Histol Cytol 61:483–492

    Article  Google Scholar 

  4. Yamamoto T, Domon T, Takahashi S et al (2007) Mineralization process during acellular cementogenesis in rat molars: a histochemical and immunohistochemical study using fresh-frozen sections. Histoch Cell Biol 127:303–311

    Article  CAS  Google Scholar 

  5. Ishimaru T, Nakazono M, Masumura T et al (2007) A method for obtaining high integrity RNA from developing aleurone cells and starchy endosperm in rice (Oryza sativa L.) by laser microdissection. Plant Sci 173:321–326

    Article  CAS  Google Scholar 

  6. Hayashi Y, Matsunaga T, Yamamoto G et al (2010) Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis. J Periodontal Res 45:489–495

    Article  Google Scholar 

  7. Zhang J, Kawashima N, Suda H et al (2006) The existence of CD11c+ sentinel and F4/80+ interstitial dendritic cells in dental pulp and their dynamics and functional properties. Int Immunol 18:1375–1384

    Article  Google Scholar 

  8. Norris Jeremy L, Caprioli Richard M (2013) Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research. Chem Rev 113(4):2309–2342

    Article  CAS  Google Scholar 

  9. Ruibing C, Limei H, Robert S et al (2009) Three dimensional mapping of neuropeptides and lipids in crustacean brain by mass spectral imaging. J Am Soc Mass Spectrom 20:1068–1077

    Article  Google Scholar 

  10. Anna N, Fehniger Thomas E, Lena G et al (2010) Fine mapping the spatial distribution and concentration of unlabeled drugs within tissue micro-compartments using imaging mass. PLoS One 5(7):e11411. Bibcode: PLoSO.511411N

    Article  Google Scholar 

  11. Watanabe H, Murata Y, Miura M et al (2006) In-vivo visualization of radiation-induced apoptosis using 125I-annexin V. Nucl Med Commun 27:81–89

    Article  CAS  Google Scholar 

  12. Tanaka M, Kawamoto T, Matsumoto H (2010) Distribution of 14C-bisphenol a in pregnant and newborn mice. Dent Mater 26:181–187

    Article  Google Scholar 

  13. Kawamoto T (1990) Light microscopic autoradiography for study of early changes in the distribution of water-soluble materials. J Histochem Cytochem 38:1805–1814

    Article  CAS  Google Scholar 

  14. Kawamoto T, Shimizu M (1997) Pathway and speed of calcium movement from blood to mineralizing enamel. J Histochem Cytochem 45:213–230

    Article  CAS  Google Scholar 

  15. Kawamoto T, Shimizu M (2000) A method for preparing 2- to 50-mm-thick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem Cell Biol 113:331–339

    Article  CAS  Google Scholar 

  16. Kawamoto T (2003) Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol 66(2):123–143

    Article  Google Scholar 

  17. Kawamoto T, Kawamoto K (2014) Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamoto’s film method (2012). Methods Mol Biol 1130:149–164

    Article  CAS  Google Scholar 

  18. Shiozuka M, Wagatsuma A, Kawamoto T et al (2010) Transdermal delivery of a readthrough-inducing drug: a new approach of gentamicin administration for the treatment of nonsense mutation-mediated disorders. J Biochem 147:463–470

    Article  CAS  Google Scholar 

  19. Hata M, Kawamoto T, Kawai M (2010) Differential expression patterns of tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor. Med Mol Morphol 43:102–106

    Article  CAS  Google Scholar 

  20. Itoh M, Kawamoto T, Tatsukawa H et al (2011) In situ detection of active transglutaminases for keratinocyte type (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides. J Histochem Cytochem 59(2):180–187

    Article  CAS  Google Scholar 

  21. Nishikawa S, Kawamoto T (2012) Planer cell polarity protein localization in the secretory ameloblasts of rat incisors. J Histochem Cytochem 60:376–385

    Article  CAS  Google Scholar 

  22. Arima Y, Harada M, Kamimura D et al (2012) Regional neural activation defines a gateway for autoreactive T cells to cross the blood-brain barrier. Cell 148(2):447–457

    Article  CAS  Google Scholar 

  23. Ushiku C, Adams DJ, Jiang X et al (2010) Long bone fracture repair in mice harboring GFP reporters for cells within the osteoblastic lineage. J Orthop Res 28:1338–1347

    Article  CAS  Google Scholar 

  24. Mina F, Katsuma K, Risa Y et al (2013) Identification of a highly reactive substrate peptide for TG6: detection of its transglutaminase activity in the skin epidermis using the peptide. FEBS J 280:1420–1429

    Article  Google Scholar 

  25. Takimoto A, Kawatsu M, Yoshimoto Y et al (2015) Scleraxis and osterix antagonistically regulate tensile force-responsive remodeling of the periodontal ligament and alveolar bone. Development 142:787–796

    Article  CAS  Google Scholar 

  26. Arima Y, Kamimura D, Atsumi T et al (2015) A pain-mediated neural signal induces relapse in multiple sclerosis models. eLife 2:e08733

    Article  Google Scholar 

  27. Nishikawa S, Kawamoto T (2015) Localization of core planar cell polarity proteins, PRICKLEs, in the ameloblasts of rat incisors. Acta Histochem Cytochem 48(2):37–45

    Article  CAS  Google Scholar 

  28. Kikuta A, Furukawa E, Ogawa R et al (2015) Biochemical characterization of medaka (Oryzias latipes) transglutaminases, OlTGK1 and OlTGK2, as orthologues of human keratinocyte-type transglutaminase (TG1). PLoS One 10(12):e0144194

    Article  Google Scholar 

  29. Wada N, Kawamoto T, Sato Y (2016) A novel application of a cryosectioning technique to undecalcified coral specimens. Mar Biol 163:1–9

    Article  CAS  Google Scholar 

  30. Arima Y, Ohki T, Nishikawa N et al (2017) Brain micro-inflammation at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit. eLife 6:e25517

    Article  Google Scholar 

  31. Chiou WY, Tsugane K, Kawamoto T et al (2018) Easy sectioning of whole grain of rice using cryomicrotome. Breed Sci 68(3):381–384

    Article  CAS  Google Scholar 

  32. Manne C, Takaya A, Yamasaki Y et al (2019) Salmonella SiiE prevents an efficient humoral immune memory by interfering with IgG+ plasma cell persistence in the bone marrow. Proc Natl Acad Sci USA 116(15):7425–7430

    Article  CAS  Google Scholar 

  33. Morodomi Y, Kanaji S, Won E et al (2019) Modified application of Kawamoto’s film method for super resolution imaging of megakaryocytes in undecalcified bone marrow. Res Pract Thromb Haemost 4:86–91. https://doi.org/10.1002/rth2.12276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yoshida M, Sato S, Kawamoto T et al (2019) Cryosection preparation for histological study, gene expression analysis and imaging mass spectrometry. J Plant Biol Res 1(1):1–7

    Google Scholar 

  35. Saigusa D, Saito R, Kawamoto K et al (2019) Conductive adhesive film expands the utility of matrix-assisted laser desorption/ionization mass spectrometry imaging. Anal Chem 91(14):8979–8986

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Radioisotope Research Institute, School of Dental Medicine, Tsurumi University, Yokohama, Japan

    Tadafumi Kawamoto & Komei Kawamoto

Authors
  1. Tadafumi Kawamoto
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  2. Komei Kawamoto
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Corresponding author

Correspondence to Tadafumi Kawamoto .

Editor information

Editors and Affiliations

  1. Department of Orthopaedic Surgery and Cell Biology, Duke Cellular, Developmental, and Genome Laboratories, Duke University School of Medicine, Durham, USA

    Matthew J. Hilton

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Kawamoto, T., Kawamoto, K. (2021). Preparation of Thin Frozen Sections from Nonfixed and Undecalcified Hard Tissues Using Kawamoto’s Film Method (2020). In: Hilton, M.J. (eds) Skeletal Development and Repair. Methods in Molecular Biology, vol 2230. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1028-2_15

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  • DOI: https://doi.org/10.1007/978-1-0716-1028-2_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1027-5

  • Online ISBN: 978-1-0716-1028-2

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