Sep 12, 2021
Epifluorescence-Microscopy-VLPs
- 1Department of Food Science, University of Copenhagen
- FOOD Micro UCPH
Document Citation: Denitsa Stefanova 2021. Epifluorescence-Microscopy-VLPs. protocols.io https://dx.doi.org/10.17504/protocols.io.bx6cpraw
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: September 12, 2021
Last Modified: November 26, 2021
Document Integer ID: 53156
Abstract
Epifluorescence Microscopy for Viral-like Particles
-Virome Studies-
Reagents
- SYBR Gold
- Glycerol
- PBS: (0.05M Na2HPO4, 0.85%(wt/vol) NaCl (pH 7.5)
- 0.02µm filtered-autoclaved MilliQwater
Equipment
- 0.02µm filter whatman Al2O3 (Whatman: 6809-6002)
- Microscope glass slides
- Glass coverslips
- Falcon tubes
- Petri dishes
- Wipes
- Swinnex Filter holder (Millipore: SX0002500)
- 5ml syringe
Reagents Setup
- 50% glycerol/50% PBS (1:1) (Anti-fading/mounting Solution)
Sterilized through 0.02µm membrane, autoclaved and stored in falcon tubes at -20°C.
- SYBR Gold solution (1000x)
975µl of 0.02µm filter-autoclaved MilliQwater
25 µl of SYBR Gold 1000x (prepared)
Prepare solution fresh-before use
Protocol
Filter Preparation
1. Filter holders (Swinnex) must be autoclaved or immersed in 70% EtOH overnight.
2. Subsequently, place the 0.02µm anodisc on top of the filter, close the lid and the filter is ready to use (REMEMBER to use filter forceps and wear gloves to avoid contamination).
Dilution of phages
3. Dilute 5 µl of CsCl gradient sample in 2.5 ml of 0.02µm filtered-autoclaved MilliQwater.
4. Using a 5 ml syringe, filter the diluted samples. When filtrating, be gentle with the pressure applied to avoid damage of the viral-like particles.
5. Carefully, remove anodisc (from holder) using filter forceps and place it on a clean Kimwipe to blot the back of the Anodisc filter.
6. Anodisc should be dried, by placing it on a new Kimwipe in a dark box or bench drawer. When the filter is dried, it will not be translucent (10-15 min).
Preparation of SYBR Gold for staining (while filters are drying)
7. Enumerate petri dishes according to your samples/treatments.
8. Dispense 100µl droplet of the SYBR Gold solution (1:38) on a Petri Dish.
9. Once anodiscs are dried, place one of them onto a 100 µl droplet and stain for 25-30 min on DARKconditions, e.g. in a dark box or bench drawer.
10 .After staining, carefully pick up the anodiscs and repeat drying procedures from steps 6 and 7.
11. Thereafter, dried anodiscs are mounted on labeled glass slides. Add 15 µl of mounting medium (ANTI-FADING solution) onto the glass to facilitate the filter positioning. Subsequently, 15 µl of mounting medium (ANTI-FADING solution) are also deposited on top of the anodisc to facilitate attachment of the glass coverslip (Noble & Fuhrman, 1998).
12. Sample is ready to be observed though the FM.