Zeiss Lightsheet Z.1, Operating Manual

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CHAPTER 2 - SAMPLE PREPARATION
Lightsheet Z.1 Sample Mounting for LSFM Carl Zeiss
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2.4 Fixation and Fixatives
Many experimental samples will require fixation prior to imaging. The goal of fixation is to maintain
cellular structure as close as possible to the native state. Proper fixation typically facilitates
immunohistochemical analyses if desired, and is an important step prior to further processing. Specialized
fixation procedures and processing may be required for certain tissues (e.g. bone de-calcification) or
preserving specific target antigens.
The processing of most samples begins with fixation to preserve morphology. A fixation method must
take into account two things: the preservation of cellular 3-D structure and maintenance of good access
to antigenic sites. The goal is to preserve sufficient cellular organization to allow identification of the
features of interest, but not to destroy the antigenicity of the target. Fixation is also frequently combined
with permeabilization to allow the staining solutions used in later steps access to the cellular interior.
Commonly used histological methods of fixation and permeabilization often consist of treating the cells
with solvents, such as methanol. While these methods are rapid-acting precipitating fixatives, they are
also good permeabilizing agents, but have one significant negative consequence: cellular shrinkage. The
degree of shrinkage may be almost insignificant for monolayers of cells, but will distort tissue samples
dramatically. To take full advantage of the three-dimensional reconstruction capability of the LSFM
microscope, the use of a fixative that does not destroy in vivo structure and organization is imperative.
It is important to remember that different specimens may require different fixation methods. Testing and
optimizing for each new sample type will ensure that the best balance between preservation and labeling
is obtained. Fixing and permeabilizing your cells affects the cell morphology and the availability of the
antigen you are trying to detect. You may get different results with different reagents, times and
concentrations, hence the need for protocol optimization. The distortion of cell morphology is something
to bear in mind when interpreting the images.
2.5 Stains and Staining
In LSFM, like in any microscopy technique using fluorescence, the sample can be labeled using specific
fluorescent dyes, fluorescent proteins or fluorescently coupled antibodies. Two basic techniques are
generally used: direct labeling and indirect labeling. Both labeling methods are suitable for LSF
microscopy. Direct labeling consists of using fluorescent proteins, fluorescently labeled primary antibody
or a dye that cause the structure of interest to become fluorescent. Advantages of this method include
speed and ease of application. A potential disadvantage is lack of sensitivity (low signal intensity). The
indirect method involves binding a primary antibody to the epitope of interest, followed by a
fluorescently labeled secondary antibody. The main advantage of using this technique is the great
amplification of signal possible through an antibody cascade. The disadvantages include increased
complexity, the method is more time consuming, and there are often problems with non-specific
antibody reactions.