Image
Left: Correct orientation, confluent monolayer. Right: Incorrect orientation, cell density too low.

Left: Correct orientation, confluent monolayer. Right: Incorrect orientation, cell density too low.

 

General tips to handle sapphire disks:

  • Disks should be glow discharged to make them hydrophilic; please coordinate with EMC to get the required amount of disks just before you seed the cells.

  • Disks are coated with a gold layer, in which the figure 2 is inscribed to keep track of the orientation; make sure the 2 faces up before and after the cells are plated on the disk.

  • Handle disks using very fine forceps: do not touch the coated surface, it will easily scratch.

  • Consider limiting the total number of sapphire disks per plate, especially when using multi-well plates, to minimize the handling time outside the incubator when freezing.

  • Disks are expensive, please return all unused disks.

Protocol

Download protocol (PDF)

  1. Sterilize disks before seeding cells:

    • Disks and forceps should be sterilized (e.g. UV light, heat, microwave).

    • Coating of disks with collagen, poly L lysine, gelatin, or similar is optional, but recommended especially with less adherent cell types.

  2. Transfer disks into medium:

    • Use wells larger than 96-well to allow easy removal of disks.

    • To prevent disks from floating: add some medium to the culture well first, then push the disk under the surface of the medium all the way to the bottom; make sure no air bubbles are attached to the disks.

    • Use two or more disks per well.

  3. Add cell suspension:

    • Ideally the cells will reach ~80% confluency by the day of HPF.

    • Ensure that the disks are oriented correctly before adding the cell suspension.

    • Cell suspension should be added very gently to avoid flipping the disks.

    • Shake well gently to spread cells evenly.

  4. Transfer to incubator:

    • Ensure that the disks are still oriented correctly before placing into incubator.

  5. If cells are frozen alive, transfer to EMC at least one day before processing:

    • Coordinate transfer with EMC to make sure the incubator is available.

    • Minimize temperature change by transporting cells in a thermo/styrofoam box during transport.

    • Keep transport times brief, and be careful not to disturb the cells, flip the disks, etc.

  6. If cells are chemically fixed before HPF:

    • Get EM grade fixative from EMC.

    • Aspirate most of the medium (it is not necessary to wash cells), carefully add warm (!!) fixative and incubate at room temperature for 30 min.

    • Carefully replace fixative with PBS.

 

Image
Image of immunolabeling

Immunogold-labeling of Histone 3 on Tokuyasu cro-section. Nucleus of muscle cell. 
Left: actin filaments and Z-bands, mitochondria (M). Right: Nucleolus showing specific lableing of Histone 3 with protein A-coated goald (10 nm, some punctate dots delineated with white arrows). 

 

Intracellular localization of proteins is of utmost importance when characterizing protein functions. The most promising approach to perform immunocytochemistry on an ultrastructural level is to do the labeling on biological material which has not been dehydrated or embedded in resin.

For immunogold-labeling we freeze cryoprotected samples and prepare thin (Tokuyasu-) cryosections from the frozen material. The labeling is then performed on thawed sections. The procedure is very similar to standard immunocytochemistry protocols for fluorescence microscopy, but we usually use protein A coated gold particles (5 nm or 10 nm) to bind to specific primary antibodies instead of flourophores. The dense gold particles are easily recognized as “immunogold” labels in the beam of the transmission electron microscope.

Keeping the sample hydrated during the entire labeling procedure preserves morphology and antigenicity extremely well, thus allowing specific immuno-localization at high resolution with great labeling success rates.

If you want to label your protein of interest please contact Martin Schauflinger at schauflingerm@missouri.edu. For more information and detailed protocols also see the following literature:

 

 

  • Individual training from an EMC staff member is required to operate any of our instruments.

  • Failure to sign into a session within 30 minutes after the scheduled starting time will enable other users to sign up for this session.

  • Cancellation of sessions are to be made 24 hours before the scheduled starting time to avoid charges.

  • Make sure no magnetic particles are inserted into the microscopes! Contact EMC staff if your samples are magnetic.