3.2 Instrument Setup
1. Replace expended Drierite (pink in color) in the drying tube
with dry Drierite (blue).
2. Turn on dehumidifier to eliminate excess moisture in the air.
3. Turn on fish pump and cooling bath (set to À6.6
C), and
allow to cool down to set temperature (see Note 4).
4. Place three blocks of dry ice in a Styrofoam box, and place the
aluminum block on top to allow it to cool for at least 15 min
before testing.
5. Once the cooling bath has reached À6.6
C, turn on the peltier
unit, and wait until it reaches À6.4
C.
6. Fit the camera into the microscope, turn on the camera, ensure
the flash is off, and set zoom to 4.8Â. Set the microscope to
20Â magnification.
3.3 IRI Sample
Testing via
Splat Assay
This method is described in [62].
1. Wipe the surface of the aluminum block using a Kimwipe.
2. Dispense 10 μL of a sample concentration (dissolved in PBS
solution) from a height of 2 m onto the polished aluminum
block cooled by dry ice (see Note 5).
3. Using a pre-cooled spatula and tweezers, carefully push the
frozen wafer formed on the aluminum block onto a glass
coverslip (see Note 6).
4. Place the coverslip containing the frozen wafer onto the cryostage, and allow it to anneal for 40 min.
5. Place the cryostage under the microscope, and take several
pictures of the ice wafer at different areas, excluding the edges.
6. Repeat for each sample concentration, including the standard
solution, in triplicate.
3.4 IRI Sample
Testing via
Sucrose Assay
This method is described in [68].
1. Wipe the surface of the aluminum block using a Kimwipe.
2. Dispense 1 μL of a sample concentration (dissolved in 40%
sucrose solution) onto a glass coverslip.
3. Place another glass coverslip on top, and apply a minimum
amount of pressure to ensure even distribution of the sandwiched solution.
4. Transfer the coverslip sandwich onto the pre-cooled aluminum
block, and allow it to cool for 1 min.
5. Remove the coverslip from the block, and transfer it onto the
cryostage for 40 min to allow annealing.
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Anna A. Ampaw et al.
1. Replace expended Drierite (pink in color) in the drying tube
with dry Drierite (blue).
2. Turn on dehumidifier to eliminate excess moisture in the air.
3. Turn on fish pump and cooling bath (set to À6.6
C), and
allow to cool down to set temperature (see Note 4).
4. Place three blocks of dry ice in a Styrofoam box, and place the
aluminum block on top to allow it to cool for at least 15 min
before testing.
5. Once the cooling bath has reached À6.6
C, turn on the peltier
unit, and wait until it reaches À6.4
C.
6. Fit the camera into the microscope, turn on the camera, ensure
the flash is off, and set zoom to 4.8Â. Set the microscope to
20Â magnification.
3.3 IRI Sample
Testing via
Splat Assay
This method is described in [62].
1. Wipe the surface of the aluminum block using a Kimwipe.
2. Dispense 10 μL of a sample concentration (dissolved in PBS
solution) from a height of 2 m onto the polished aluminum
block cooled by dry ice (see Note 5).
3. Using a pre-cooled spatula and tweezers, carefully push the
frozen wafer formed on the aluminum block onto a glass
coverslip (see Note 6).
4. Place the coverslip containing the frozen wafer onto the cryostage, and allow it to anneal for 40 min.
5. Place the cryostage under the microscope, and take several
pictures of the ice wafer at different areas, excluding the edges.
6. Repeat for each sample concentration, including the standard
solution, in triplicate.
3.4 IRI Sample
Testing via
Sucrose Assay
This method is described in [68].
1. Wipe the surface of the aluminum block using a Kimwipe.
2. Dispense 1 μL of a sample concentration (dissolved in 40%
sucrose solution) onto a glass coverslip.
3. Place another glass coverslip on top, and apply a minimum
amount of pressure to ensure even distribution of the sandwiched solution.
4. Transfer the coverslip sandwich onto the pre-cooled aluminum
block, and allow it to cool for 1 min.
5. Remove the coverslip from the block, and transfer it onto the
cryostage for 40 min to allow annealing.
278
Anna A. Ampaw et al.