Confocal laser scanning microscopy on Buttermilk

Author: Louise Krebs / Alexandre Bastien
Date: 2021-12-02

1. Dilute Fast Green in distilled water at a concentration of 1 mg/mL
2. Dilute Nile Red in acetone at a concentration of 1 mg/mL
3. Mix 100 µL sample with 6 µL Fast Green and 2 µL Nile Red
4. Rest for 20 min

1. Dilute low-melting agarose in distilled water at a concentration of 3 % (p/v)
2. On a hot plate, melt the agarose-water-mix until agarose is completely dissolved while constantly stirring it
3. On a hot plate, keep solution at 40 °C

1. Carefully clean the microscopy plates with methanol and Kimwipes
2. Attach double-side tape on the plates (see picture) and place them on the hot plate (40 °C)

1. Place additional clean microscopy plates on the hot plate
2. On these plates, mix 40 µL sample with 40 µL agarose and mix fast, but properly (don’t wait too long to avoid evaporation!)
3. Take 10 µL of the sample-agarose-mixture and place it between the double-side tape on the microscopy plate
4. Quickly place the coverslip on top and press slightly
5. Remove the microscopy plate from the hot plate
6. After 5 minute waiting time, use nailpolish (colorless, fast-drying) to seal the edges of the coverslip in order to avoid evaporation of the sample

1. Analyze plates with confocal laser scanning microscopy
2. Fast Green: HeNe laser at 633 nm, filter BA 655-755
3. Nile Red: Ar+ laser at 488 nm