Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONCarl Zeiss Specific Examples of Sample Preparation Lightsheet Z.134 000000-1790-528 02/2013If cells are grown in a different manner it is possible to mix them with a supporting gel prior toloading into a chamber. They can also be grown within the gel already present in an incubationchamber. This limits damage, shear and temperature changes during sample preparation andhandling.5. The agarose incubation chamber is mounted on a specific holder. The polymer chamber can beeither clipped or glued to a supportive holder.Eukaryotic cells are highly sensitive to environmental change (temperature, pH, osmotic pressureetc.). 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 andpre-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 CystsImmunofluorescence allows highlighting of specific proteins or structures using specific antibodies. Thisprotocol is used to perform immunofluorescence on cysts which are three-dimensional cell structures thatcan 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 blockMethod1. MDCK cell cysts grown in extracellular matrix are collected and centrifuged at 500-1000g to pelletthe cysts with the gel.2. The supernatant is removed and replaced with 4 % paraformaldehyde and incubated for15 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 theparaformaldehyde, and then incubated for 10 minutes.4. The gel pellet is washed twice with PBS (500-1000 g, 5 minutes).