1992 Fiscal Year Final Research Report Summary
Development of absolute quantitative immunoelectron microscopy
| Project/Area Number |
03557004
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | Kansai Medical University |
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Principal Investigator |
TASHIRO Yutaka Kansai Medical University President, 学長 (40077558)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIMORI Tamotsu Kansai Medical University Assistant, 医学部, 助手 (60191649)
YAMAMOTO Akitsugu Kansai Medical University Lecturer, 医学部, 講師 (30174775)
MASAKI Ryuichi Kansai Medical University Lecturer, 医学部, 講師 (70140283)
OMORI Koichiro Kansai Medical University Associate Professor, 医学部, 助教授 (80094465)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Absolute quantitative immunoelectron microscopy / Colloidal gold immunoelectron microscopy / Immunoblot method / Cytochrome P-450 HB / Na,K-ATPase / Lysosomal pH / DAMP method |
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| Research Abstract |
In this research project, we intended to develop a quantitative immunoelectron microscopic method which makes it possible to estimate the absolute number of antigen molecules on ultrathin sections by protein A-colloidal gold technique. For this purpose enough antibody should be added to carry out immunological reaction at a saturation level of antibody and all the experimental conditions should be same.
We have tested validity of our method by three experiments.
1) Quantitative analysis of Na, K-ATPase along rat nephron. Ultrastructural localization of Na, K-ATPase along rat nephron segments was investigated quantitatively of immunogold electron microscopy. The relative number of gold particles/mum nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na,K-ATPase activity profile in rat nephron. which was determined bio
… More
chemically with a microenzymatic method.
2) Increase in lysosomal pH after treatment with bafilomycin was estimated quantitatively by immunogold DAMP method. and the results obtained were in good agreement with those estimated by the light microscopic FITC-dextran method.
3) We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P-450(P450HB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. We counted the number of gold particles per mum of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450HB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P-450HB,indicating good correlation of the two variables. Thus. the induction of cytochrome P-450HB can be quantitatively and reliably investigated by immunogold electron microscopy. (J. Histochem. Cytochem. 40:73-82,1992) Less
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