2015 Fiscal Year Annual Research Report
| Project/Area Number |
26840049
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| Research Institution | Kyoto University |
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Principal Investigator |
PUJALS Silvia 京都大学, 物質-細胞統合システム拠点, 研究員 (10611866)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Keywords | Bioimaging / Electron microscopy |
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| Outline of Annual Research Achievements |
During this year we have applied SNAP tag method to achieve the electron microscopy labeling for basal membrane sheets and intracellular proteins. We have used Caveolin-1 protein, as a model, as it has been widely studied in our lab.
First, we successfully cloned Caveolin-1 gene into the SNAP-tag plasmid. Then we checked it by confocal microscopy (CLSM), both with live and fixed cells. For those experiments we used either BG-OregonGreen or BG-Biotin, with a further step of Streptavidin-AF488. In the first case we could observe the cells either live or fixed. In the case of using the biotin-streptavidin we had to work with fixed and permeabilised cells or with unroofed cells. We checked the cells by CLSM and there was a nice punctuated labeling, characteristic of caveolae.
We took the next steps and performed electron microscopy (EM). The EM technique that we use is called quick freeze, deep etch EM, which is able to freeze cells instantly and preserve the cellular structures intact. Then after etching the sample and rotatory evaporating Pt and C over it, it is ready for transmission EM (TEM) observation.
Thus,we assayed the SNAP tag system for the labeling of basal membrane sheets. After quick-freezing and freeze-drying the cells, we observed them by TEM to confirm the specific labeling of Caveolin-1 in caveolae. We had to find the optimal conditions for the labeling of unroofed cells (incubation time, concentration of BG-biotin, nanoparticle-streptavidin, etc.). The most difficult part was to find a good nanoparticle-streptavidin conjugate with electron microscope quality.
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