TY - JOUR
T1 - Three-dimensional reconstruction of living mouse liver tissues using cryotechniques with confocal laser scanning microscopy
AU - Saitoh, Yurika
AU - Terada, Nobuo
AU - Saitoh, Sei
AU - Ohno, Nobuhiko
AU - Fujii, Yasuhisa
AU - Ohno, Shinichi
N1 - Funding Information:
Grant for young investigators from the University of Yamanashi (to Y.S.); Grant (no. 861600070) for innovative research from the Japan Science and Technology Agency (to S.O.).
PY - 2010/12
Y1 - 2010/12
N2 - Soluble proteins and glycogen particles are well preserved in paraffin-embedded sections prepared by in vivo cryotechnique (IVCT) and cryobiopsy followed by freeze substitution fixation. We performed confocal laser scanning microscopic analyses on the distributions of glycogen with periodic acid-Schiff (PAS) staining and serum proteins with immunostaining for mouse liver tissues. Livers of fully fed mice showed a strong fluorescence signal of PAS staining in all hepatocytes and immunofluorescence of immunoglobulin kappa light chain (Igκ) in blood vessels and bile canaliculi. However, some hepatocytes in mechanically damaged livers were PAS-negative and Igκ-immunopositive, showing extraction of glycogen particles and infiltration of serum proteins in hepatocytes. By three-dimensional (3D) reconstruction of serial optical sections, interconnecting hepatic sinusoids and bile canaliculi were detected with Igκ immunostaining between trabecular hepatocytes that were PAS stained. In PAS-stained samples under fasting conditions, interstitial structures along sinusoids were clarified in vivo by 3D reconstruction because of the lower PAS staining intensity of hepatocytes. In addition, 100-μm-thick eosin-stained slices provided 3D structural images more than 30 μm in thickness away from tissue surfaces, showing blood vessels with flowing erythrocytes and networks of bile ducts and canaliculi. IVCT and cryobiopsy with histochemical analyses enabled us to visualize native hepatocytic glycogen and 3D structures, such as vascular networks, reflecting their living states by confocal laser scanning microscopy.
AB - Soluble proteins and glycogen particles are well preserved in paraffin-embedded sections prepared by in vivo cryotechnique (IVCT) and cryobiopsy followed by freeze substitution fixation. We performed confocal laser scanning microscopic analyses on the distributions of glycogen with periodic acid-Schiff (PAS) staining and serum proteins with immunostaining for mouse liver tissues. Livers of fully fed mice showed a strong fluorescence signal of PAS staining in all hepatocytes and immunofluorescence of immunoglobulin kappa light chain (Igκ) in blood vessels and bile canaliculi. However, some hepatocytes in mechanically damaged livers were PAS-negative and Igκ-immunopositive, showing extraction of glycogen particles and infiltration of serum proteins in hepatocytes. By three-dimensional (3D) reconstruction of serial optical sections, interconnecting hepatic sinusoids and bile canaliculi were detected with Igκ immunostaining between trabecular hepatocytes that were PAS stained. In PAS-stained samples under fasting conditions, interstitial structures along sinusoids were clarified in vivo by 3D reconstruction because of the lower PAS staining intensity of hepatocytes. In addition, 100-μm-thick eosin-stained slices provided 3D structural images more than 30 μm in thickness away from tissue surfaces, showing blood vessels with flowing erythrocytes and networks of bile ducts and canaliculi. IVCT and cryobiopsy with histochemical analyses enabled us to visualize native hepatocytic glycogen and 3D structures, such as vascular networks, reflecting their living states by confocal laser scanning microscopy.
UR - http://www.scopus.com/inward/record.url?scp=78649584852&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78649584852&partnerID=8YFLogxK
U2 - 10.1093/jmicro/dfq065
DO - 10.1093/jmicro/dfq065
M3 - Article
C2 - 20709827
AN - SCOPUS:78649584852
SN - 0022-0744
VL - 59
SP - 513
EP - 525
JO - Journal of Electron Microscopy
JF - Journal of Electron Microscopy
IS - 6
ER -