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CHAPTER 2 - SAMPLE PREPARATION
Carl Zeiss
Specific Examples of Sample Preparation
Lightsheet Z.1
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02/2013
If cells are grown in a different manner it is possible to mix them with a supporting gel prior to
loading into a chamber. They can also be grown within the gel already present in an incubation
chamber. This limits damage, shear and temperature changes during sample preparation and
handling.
5.
The agarose incubation chamber is mounted on a specific holder. The polymer chamber can be
either clipped or glued to a supportive holder.
Eukaryotic cells are highly sensitive to environmental change (temperature, pH, osmotic pressure
etc.). The transfer steps must be rapid and carried out in a sterile manner (wherever possible)
especially for long term time lapse experiments. It is important to be gentle and use cut tips and
pre-warmed materials at all times, including the sample chamber.
6.
Monitor the cell status during imaging to check viability and changes.
3.6
Immunostaining and Preparation of MDCK Cell Cysts
Immunofluorescence allows highlighting of specific proteins or structures using specific antibodies. This
protocol is used to perform immunofluorescence on cysts which are three-dimensional cell structures that
can be grown in extracellular matrix gel such as collagen.
Equipment and reagents
−
1.5 % Low Melting Point (LMP) agarose in water or PBS
−
Capillary (Size 4, Blue, #701999, BRAND GmbH)
−
Electrical thread (1.6 mm) or plunger
−
4 % paraformaldehyde solution
−
Antibodies (primary and secondary)
−
PBS
−
Triton X-100
−
Bovine Serum Albumin (BSA) or Foetal Calf Serum (FCS)
−
Heating block
Method
1.
MDCK cell cysts grown in extracellular matrix are collected and centrifuged at 500-1000g to pellet
the cysts with the gel.
2.
The supernatant is removed and replaced with 4 % paraformaldehyde and incubated for
15 minutes on a wheel or rocker to efficiently mix the gel pellet within the fixative.
3.
The gel is pelleted and the supernatant is replaced by 0.1M glycine to quench the
paraformaldehyde, and then incubated for 10 minutes.
4.
The gel pellet is washed twice with PBS (500-1000 g, 5 minutes).
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