en:protocoles:confocal_on_soft_matrices
Confocal laser scanning microscopy on Buttermilk
Author: Louise Krebs / Alexandre Bastien
Date: 2021-12-02
1. Sample coloring
- Dilute Fast Green in distilled water at a concentration of 1 mg/mL
- Dilute Nile Red in acetone at a concentration of 1 mg/mL
- Mix 100 µL sample with 6 µL Fast Green and 2 µL Nile Red
- Rest for 20 min
2. Agarose preparation
- Dilute low-melting agarose in distilled water at a concentration of 3 % (p/v)
- On a hot plate, melt the agarose-water-mix until agarose is completely dissolved while constantly stirring it
- On a hot plate, keep solution at 40 °C
3. Microscopy plate preparation
- Carefully clean the microscopy plates with methanol and Kimwipes
- Attach double-side tape on the plates (see picture) and place them on the hot plate (40 °C)
4. Sample preparation
- Place additional clean microscopy plates on the hot plate
- On these plates, mix 40 µL sample with 40 µL agarose and mix fast, but properly (don’t wait too long to avoid evaporation!)
- Take 10 µL of the sample-agarose-mixture and place it between the double-side tape on the microscopy plate
- Quickly place the coverslip on top and press slightly
- Remove the microscopy plate from the hot plate
- 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
5. Imaging
- Analyze plates with confocal laser scanning microscopy
- Fast Green: HeNe laser at 633 nm, filter BA 655-755
- Nile Red: Ar+ laser at 488 nm
en/protocoles/confocal_on_soft_matrices.txt · Last modified: 2022/05/24 13:30 by Administrateur