Methods and protocols
Immunofluorescence microscopy: The location of proteins of interest within cardiomyocytes is investigated by labelling cultured cells or cryosections of heart with specific antibodies whose position can be revealed with fluorescent markers. Thin cryosections are obtained from lightly fixed and cryoprotected heart tissue.
A number of different kinds of samples are examined in the electron microscope to obtain details of structure and protein organisation at the molecular level. The transmission electron microscopes and other specialist apparatus are housed in the college’s Centre for Ultrastructural Imaging (CUI) on the Guy’s Campus.
- Immunogold techniques: Antibodies in thin cryosections as above are further labelled with molecules complexed to small gold particles, 5nm to 20nm in diameter. These can be seen in the electron microscope and localised with respect to structural features of the myocytes.
- Conventional imaging. Normal and diseased hearts are fixed and embedded by conventional means. Thin sections are examined in the transmission electron microscope and the structures are compared. From the images, measurements such as relative mitochondrial volume and dimensions of certain features are obtained.
- Frozen specimens: To improve the specimen preservation we have been using high pressure freezing to maintain the structure and obviate any preparative artefacts. The samples are then freeze substituted and embedded for electron microscopy.
To understand the intricate convolutions of the membrane and its relationship to associated structures requires three dimensional imaging. This can be done by tomographic reconstruction: A series of pictures of tilted views of the specimen are obtained and recombined computationally to reveal the three dimensional organisation. The electron microscope used is the high voltage Technai 20 in the CUI. Some 140 images at 1o tilt intervals about two perpendicular axes are used for the reconstruction. Commercial and public domain software is used for image analysis.