TITLE: Fluorescence microscopy with the marine heterotrophic flagellate Cafeteria roenbergensis
AUTHORS: Monica Berjon-Otero, Sarah Duponchel, Matthias Fischer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Fluorescence microscopy is essential to understand, between others, the cell organization and the life cycle of viruses that infect them. This protocol describes the steps neccesary to fix and permeabilize </span><span style = "font-style:italic;">Cafeteria roenbergensis</span><span> cells allowing its staining with antibodies or chemical reactions as Click-IT reactions.</span></div></div>

[STEPS]
?. [Remove bacteria from Cafeteria roenbergensis culture]
Follow the protocol: "Lysozyme-based removal of bacteria from cultures of the marine heterotrophic flagellate Cafeteria roenbergensis"
If the culture is to be treated with other reagents, for example L-Azidohomoalanine, dilute the cells without bacteria to the required density and apply the treatment.
?. [Remove bacteria from Cafeteria roenbergensis culture]
Aliquot 5 x 106 Cafeteria cells per 1.5 ml microfuge tube for each microscopy sample
?. [Fixation and permeabilization]
Centrifuge for 5 min at 5,000 g, 20ºC
?. [Fixation and permeabilization]
Resuspend each pellet in 500 µL of 4% paraformaldehyde (PFA) in 1X PBS
?. [Fixation and permeabilization]
Incubate for 20 min at 20ºC
?. [Fixation and permeabilization]
Centrifuge for 5 min at 7,000 g, 20ºC
You can keep the sample overnight at 4 ºC by resuspending the pellet with 250 µL 1X PBSOn the following day, centrifuge for 5 min at 5,000 g, 20ºC and continue with the next step
?. [Fixation and permeabilization]
Resuspend the pellets in 500 µL of 0.5% Triton in 1X PBS
?. [Fixation and permeabilization]
Incubate for 15 min at 20ºC
?. [Filters preparation to Cafeteria immobilization]
Clean the filter-support grids of a vacuum manifold system with pure water (e.g. ELGA, milliQ)
?. [Filters preparation to Cafeteria immobilization]
Apply vaccum to remove the drop of water
?. [Filters preparation to Cafeteria immobilization]
Add a drop of ELGA water on each grid of the vacuum system that you want to use and place a 0.45 µm Millipore support filter on top of it
?. [Filters preparation to Cafeteria immobilization]
Add another drop of ELGA water on top of the Millipore filter
?. [Filters preparation to Cafeteria immobilization]
Place a 0.2 µm, 25 mm Anodisc Whatman filter on top of the ELGA water drop
?. [Filters preparation to Cafeteria immobilization]
Apply vacuum with a pressure between 5-10 Hg to remove the water
?. [Filters preparation to Cafeteria immobilization]
If neccesary, block or wash the filter with the required solution.Remove the solution by applying vacuum
?. [Filters preparation to Cafeteria immobilization]
Place the Cafeteria sample from step 8 on top of the Whatman filter
?. [Filters preparation to Cafeteria immobilization]
Apply vacuum with a pressure between 5-10 Hg to remove the permeabilization buffer and to immobilize the cells on the filter
Perform all the neccesary blocking and washing steps following the same instructions
?. [Sample staining]
Place the filter with the Cafeteria sample on a piece of Parafilm. Add the staining solution of interest on top of the filter and incubate following the manufacturer's instructions
?. [Sample staining]
Place the filter on the vacuum system again
?. [Sample staining]
Apply vacuum with a pressure between 5-10 Hg to remove the remaining staining buffer
?. [Sample staining]
If required, wash the filter again with the buffer of interest
?. [Sample staining]
If desired, stain DNA by incubating with an appropriate reagent (DAPI, SYBR, Hoechst)
?. [Sample staining]
Apply vacuum with a pressure between 5-10 Hg to remove the remaining liquid
?. [Sample mounting]
Add 10 µL of 70% glycerol on a glass slide and place the filter on top of the glycerol
The filter must be placed with the Cafeteria cells in the top!
?. [Sample mounting]
Add 10 µL of 70% glycerol on the coverslip and place it on top of the filter
?. [Sample mounting]
Apply pressure on the sample during 20 min at room temperature
?. [Sample mounting]
Seal the coverslip with nail polish
Samples can be kept at 4 °C or analyzed with the microscope immediatly