Abstract
In this chapter, we report a protocol to perform correlative light electron microscopy (CLEM) on adult Caenorhabditis elegans. We use a specific fixation protocol, which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins and, subsequently, with transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows determining the subcellular localization of the fluorescent protein.
We used CLEM to characterize the subcellular localization of the C. elegans ESCRT-II component VPS-36. VPS-36 protein localization in C. elegans muscle cell is strongly correlated with the sarcoplasmic reticulum network. Together with genetic evidences, the CLEM data support a role for ESCRT-II proteins in sarcoplasmic reticulum membrane shaping.
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Key words
- ESCRT-II
- Freeze substitution
- GMA resin
- Green fluorescent protein
- High pressure freezing
- Electron microscopy
- Muscle
- Sarcoplasmic reticulum
- VPS-36
1 Introduction
The C. elegans body-wall muscle cells (BWM) are functionally and structurally similar to mammalian striated muscle [1]. In BWM, the sarcoplasmic reticulum (SR) forms a network of thin tubules localized in close association with the contractile apparatus. It is regularly localized around the dense body (DB, the actin attachment structures corresponding to both vertebrates Z discs and costameres) and below the contractile filaments adjacent to the apical membrane between DB and M-line (Fig. 1). The SR has to maintain a strong association with the DB and the muscle apical membrane, which resists the mechanical stress associated with the repeated contractile cycles. However, mechanisms and proteins involved both in establishing and maintaining the SR network are largely unknown.
We investigated the role of ESCRT-II proteins on C. elegans sarcoplasmic reticulum structure and integrity. The endosomal sorting complexes required for transport (ESCRT 0 to III) have been initially discovered as key protein complexes involved in endosome maturation. During this process, the four ESCRT complexes, ESCRT-0, -I, -II, and -III, are sequentially recruited to the endosome membrane, allowing the sorting of ubiquitylated membrane proteins into intraluminal vesicles during multivesicular bodies maturation (MVB) (Fig. 2). Beyond this role in MVB formation, ESCRTs complexes are also involved in numerous cellular processes that involve membrane remodelling [2,3,4]. We have been investigating the ESCRT functions during Caenorhabditis elegans development and reported that ESCRT mutant phenotypes are heterogeneous [5, 6]. The observation that ESCRT-II mutants have late developmental defects, compared to that of other ESCRTs in C. elegans [7], supports a hypothesis of a specific role for ESCRT-II components. ESCRT-II is a hetero-tetramer made of VPS-22, VPS-36, and two VPS-25 subunits.
Using a combination of imaging and genetic approaches, we discovered that ESCRT-II, but neither ESCRT-0, ESCRT-I, nor ESCRT-III complexes, localizes to the sarcoplasmic reticulum, and that the depletion of ESCRT-II components results in alterations of sarcoplasmic reticulum shape. We also demonstrated that ESCRT-II mutant worms present a progressive alteration in locomotion during larval development [8].
We use Correlative Light and Electron Microscopy (CLEM) on C. elegans adults to characterize the subcellular localization of GFP-tagged VPS-36 protein in C. elegans muscle cells and showed it is closely associated to the DB and the apical membrane, which corresponds to the SR network organization. We report here the detailed protocol used to perform CLEM, which is subdivided in four main steps: (1) cryofixation of animal specimen by high pressure freezing to preserve structures; (2) freeze substitution and inclusion in GMA resin to preserve GFP fluorescence; (3) ultramicrotomy and successive observations of the sections by light and electron microscopies, and (4) correlation of fluorescent and electron microscopy images.
This protocol allows performing an easy and rapid correlation between the electron and the light microscopy, based on the dual imaging of thin sections. Although the CLEM pictures have a lower contrast than classical transmission electron microscopy [9], this approach circumvents the difficulty of the retrospective location of the regions of interest, which necessitates very specific approaches [10].
2 Materials
2.1 Caenorhabditis elegans Strains and Culture
The conditions for culturing nematode strains are very classical and they will not be detailed here [11].
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1.
Strains: RD112 (N2; Ex[vps-36::gfp;rol-6(su1006)]).
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2.
Nematode Growth Medium (NGM): dissolve 1.5 g of NaCl, 1.5 g of bactopeptone, and 0.5 ml of cholesterol (5 mg/ml) in 485 ml of water and autoclave. Cool down the medium to 55 °C and supplement it with 0.5 ml CaCl2 (1 mol/l), 0.5 ml MgSO4 (1 mol/l), 10 ml KH2PO4 (1 mol/l), and 1.65 ml KH2PO4 (1 mol/l). Pour the medium in Petri dishes (either 90 mm or 50 mm diameter plates).
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3.
Escherichia coli OP50 strain (available at the Caenorhabditis Genetic Center https://cgc.umn.edu/). Seed NGM plates with 100 μl of OP50 culture in exponential growth phase.
2.2 Cryofixation
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1.
HPF apparatus EM PACT2 (Leica Microsystem) using liquid nitrogen for cryogenic conditions and methyl cyclohexane to generate pressure.
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2.
M9 buffer: Dissolve 6 g of Na2HPO4, 3 g of KH2PO4, and 5 g of NaCl in 1 L of water, autoclave and aliquot by 200 ml. Supplement 200 ml aliquots with 200 μl of 1 M MgSO4.
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3.
M9 buffer containing 20% (w/v) Bovine Serum Albumin (BSA), store at 4 °C after complete dissolution.
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4.
1% phosphatidylcholine (sigma-aldrich) pre-coated 200 μm deep flat carriers (Leica Microsystem) (Fig. 3a). Prepare extemporaneously.
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5.
4% (w/v) low gelling agarose in 1 ml aliquots in small glass tubes, store at 4 °C.
2.3 Freeze Substitution and Resin Inclusion
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1.
Freeze substitution is performed with Automated Freeze substitution System (AFS2) with integrated binocular lens and incubating chamber (Leica Microsystem) (Fig. 3d).
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Pasteur pipettes.
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Flat embedding insert (sample container containing a reagent tray and a continuous flow ring) (Leica Microsystem) (Fig. 3b).
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Aclar sheet (Leica Microsystem).
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Glass bottle of 10 ml.
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Tweezers and « worm pick » with platinum wire (Fig. 3e, g).
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7.
Specimen processing metal block (Leica Microsystem) (Fig. 3c).
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8.
Freeze substitution buffer: 0.1% (w/v) KMnO4; 5% (v/v) water; 95% (v/v) anhydride acetone; 0.01% (v/v) osmium tetroxide (stock 4% w/v in water).
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9.
95% (v/v) acetone in water.
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10.
95% (v/v) ethanol in water.
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11.
5% uranyl acetate (w/v) in anhydride ethanol. Prepare extemporaneously, vortex, and filter (0.2 μm) the solution.
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12.
0.1% (v/v) uranyl acetate in 95% acetone. Prepare extemporaneously.
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13.
Glycol methalacrylate (GMA) acrylic resin: 68.1 ml glycol methacrylate supplemented with 30 ml of butyl methacrylate, 3 ml of H2O, and 600 mg of benzoyl peroxide. Place the tube on a rotator and rotate slowly for 1 h at 4 °C and store at −20 °C in aliquot of 10 ml.
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14.
30% (v/v) GMA in 95% ethanol. Prepare extemporaneously.
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15.
70% (v/v) GMA in 95% ethanol. Prepare extemporaneously.
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16.
0.32% (v/v) N,N-dimethyl-p-toluidine (accelerator polymerization agent) (Merck) in GMA acrylic resin. Prepare extemporaneously.
2.4 Ultramicrotomy
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1.
Ultramicrotome EM UC7 (Leica Microsystems).
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2.
Razor blade.
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3.
Glass knife.
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Diamond knife for histology (Diatome, Histo, 2 mm, 45°).
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Toluidine blue: 1% (w/v) toluidine blue and 1% (w/v) borate sodium in water. Filter (0.2 μm).
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6.
Standard light stereomicroscope.
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7.
200 mesh copper formvar carbon-coated finder grid.
2.5 Light Microscopy
We used either upright or inverted microscopes.
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1.
Inverted microscope: AxioOberver Z1 microscope (Zeiss) equipped with Evolve EMCCD camera (Roper Scientific).
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Upright microscope: Axioskop 2 Plus microscope (Zeiss) equipped with Nomarski optics coupled to a CoolSNAP camera (Roper Scientific).
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3.
35 mm cell view, a cell culture dish with glass bottom (Greiner Bio-One n°627861).
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4.
Glass slides and coverslips (22 mm × 22 mm, thickness 0.13–0.17 mm).
2.6 Transmission Electron Microscopy
JEOL1400 transmission electron microscope operating at 80 kV coupled with a Gatan 11 Mpixels SC1000 Orius CCD camera.
2.7 Correlation Analysis
Treatment and superimposition of light and electron microscopy pictures are performed with ImageJ, PowerPoint, and Photoshop softwares.
3 Methods
3.1 Cryofixation
The current protocol is designed for the analysis of adults, but it can be used for larvae and embryos upon adaptation of the nematode culture.
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1.
Grow worms on NGM plate seeded with OP50 at 20 °C.
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2.
Transfer 20–30 adults with the worm pick, into 1% phosphatidylcholine pre-coated 200 μm deep flat carrier (see Note 1) containing 5 μl of 20% BSA in M9 (Fig. 3e). Eventually, before freezing, add a drop of agarose low gelling 4% to seal the worm and the cups (see Note 2).
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3.
Put 200 μm deep flat carriers containing adults in High Pressure Freezing apparatus (HPF) and cryofix according to manual protocol using automatic mode.
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4.
Transfer flat carriers to cryogenics tubes and store in liquid nitrogen container, where they can be kept several months. During the whole process, keep flat carriers in liquid nitrogen.
3.2 Freeze Substitution and Resin Inclusion
Freeze substitution process has been adapted from previously published protocols in C. elegans or plants [12,13,14].
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1.
Day 1, put the specimen processing metal block containing the flat embedding insert in the incubating chamber of AFS2 preset at −90 °C. Using a Pasteur pipette, fill delicately the flat embedding insert with about 1.5 ml of freeze substitution buffer (Fig. 3f). Perform all subsequent steps of buffer removal or washes this way.
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2.
After precooling the AFS2 for 2 h at −90 °C, remove flat carrier from cryogenic tubes using tweezers and put them in the specimen processing metal block without defrosting the samples (see Note 3) (Fig. 3g).
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3.
Transfer a single flat carrier from cryogenics tubes in each compartment of the flat embedding insert (Fig. 3h).
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Close AFS and start the AFS program (Fig. 4). Keep samples at −90 °C for at least 5 h and up to 30 h, if convenient.
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5.
Set a temperature slope from −90 °C to −50 °C with a gradient of 5 °C per hour (Fig. 4).
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6.
Day 2, prepare 95% acetone and put in the AFS2 incubating chamber to allow temperature equilibrium for at least 15 min.
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7.
Remove the freeze substitution medium from the flat embedding insert and add 95% acetone. Incubate for 10 min and repeat the operation four times. Maintain AFS2 at −50 °C for 1 h (Fig. 4).
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8.
Prepare the 5% acetate uranyl and the 0.1% acetate uranyl in acetone and put it in the AFS2 incubating chamber to allow temperature equilibrium for at least 15 min (−50 °C).
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9.
Remove the 95% acetone solution and add 0.1% acetate uranyl in acetone solution.
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10.
Set a temperature slope from −50 °C to −30 °C with a gradient of 5 °C per hour and program AFS to maintain temperature at −30 °C for the next 72 h (Fig. 4), but proceed to step 11 when temperature has reached −45 °C.
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11.
Remove acetone 0.1% acetate uranyl and add acetone 95%. Incubate for 10 min. Repeat the acetone washes four times. Then keep samples in the AFS incubating room for 2 h until it reaches −30 °C.
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12.
Prepare the 95% ethanol and put in the AFS2 incubating chamber for temperature equilibrium for at least 15 min (−30 °C). Remove 95% acetone and add 95% ethanol for 15 min. Repeat ethanol washes four times.
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13.
Prepare 30% and 70% GMA in 95% ethanol and store in a −20 °C freezer until using.
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14.
Remove 95% ethanol from the flat embedding insert and add 30% GMA for a 2 h incubation.
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15.
Remove 30% GMA, add 70% GMA for a 3 h incubation.
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16.
Remove 70% GMA and add 100% GMA for an overnight incubation.
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17.
Day 3, remove 100% GMA and add fresh 100% GMA for 2.5 h.
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18.
Repeat step 17.
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19.
Remove sample from the deep flat carrier and the deep flat carrier from the flat embedding insert, using integrated binocular lens and curved metallic spike and tweezers. Remove very carefully 100% GMA and incubate in fresh 100% GMA for 15 min.
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20.
Prepare 100% GMA with accelerator.
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21.
Put an aclar ring on the flat embedding insert.
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22.
Remove 100% GMA and wash with 100% GMA containing the accelerator polymerization agent.
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23.
Remove immediately the solution and add 100% GMA with the accelerator polymerization agent for an overnight incubation. Avoid overflow of each compartment.
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24.
Day 4, switch off the AFS, take off the flat embedding insert, and unmold the blocks of resin that have polymerized in each compartment. Conserve blocks at −20 °C in darkness in a vacuum-sealed freezer bag.
3.3 Ultramicrotomy
Thin sectioning follows standard protocol. The main steps are indicated below without details (Leica Microsystems EM UC7 user manual) (see Note 4).
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1.
Make a pyramid shape structure in the block around the sample with razor blade.
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2.
Smooth pyramid surface with a glass knife (speed 10 feed 500 nm) (see Note 5).
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3.
Make semi-thin sections using histological diamond knife (speed 5 feed 200 nm).
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4.
Collect semi-thin sections on a glass slide and stain them with toluidine blue.
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5.
Look at stained semi-thin sections under a classical microscope.
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6.
If the sample is correct, make semi-thin sections at slower speed (speed 2 feed 200 nm) and collect them on 200 mesh copper formvar carbon-coated finder grid. Keep the grid in darkness and at 4 °C until microscopic observations (see Note 6).
3.4 Fluorescent and Electronic Microscopy
3.4.1 Fluorescent Microscopy Using an Upright Microscope (Fig. 5a and b)
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1.
Put the grid on a glass slide with semi-thin sections on the top side.
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Add 10–20 μl of water on grid and add coverslip (see Note 7).
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After bright field and fluorescent observations, remove the coverslip and collect gently the grid with tweezers (see Note 8).
3.4.2 Fluorescent Microscopy Using Inversed Microscope (Fig. 5c and d)
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1.
Take a 35 mm cell view.
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In the middle of the glass bottom put a drop of water (5 μl).
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Put the grid upside-down on the water drop using tweezers. The water should just make a link between grid and glass slide, and the grid should not move (see Note 9).
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4.
Take bright field and fluorescent images.
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5.
Remove the grid after observation, by gently adding 1 ml of water in the dish and using tweezers.
3.4.3 Electron Microscopy
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1.
Sections are observed with a JEOL 1400 TEM instrument operating at 80 kV, using a standard procedure.
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2.
Fill the anticontamination device with liquid nitrogen, Switch on the high tension and increase it to 80 kV, with the condenser aperture inserted.
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3.
Mount grids on the specimen holder and insert it in the microscope. Turn the filament on.
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4.
For each grid, localize the areas of interest using the letter and the number identified during light microscopy on 200 mesh copper formvar carbon-coated finder grid.
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5.
Image areas of interest at low magnification and then go to higher magnification.
3.5 Correlation Between Fluorescent and Electronic Images
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1.
Perform composite images of bright field and fluorescent images with NIH ImageJ Software (https://imagej.nih.gov/ij/) (Fig. 6a).
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2.
Import the composite image in a PowerPoint file. Outline the area of interest manually (see Note 10) and report this draw on transmission electron microscopy images to put them at the same size (Fig. 6b).
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3.
Open the composite image, of bright field and fluorescent images, in Photoshop (64 Bit).
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4.
Select the image or use a selection tool to specifically choose one region of interest.
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5.
Copy the image or the selected area; create a new file with a transparent background.
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6.
Paste the image in the new file, select the magic wand tool, and click on to select the background of the image. Use the delete command to suppress the background and to obtain an image with a transparent background (Fig. 6c).
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7.
Save the file in Portable Network Graphics format (PNG) and superimpose manually using powerpoint, the PNG image, and the composite image to obtain images with identical size. Then, superimpose in a similar way the corrected-size PNG image and the electron microscopy image (Fig. 6d) (see Note 11).
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8.
Repeat steps 1–7 on higher magnification electron microscopy images to correlate ultrastructure and fluorescent signals (Fig. 6e).
4 Notes
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1.
Under a fume hood, put the 200 μm deep flat carriers in crystallizer containing 1% phosphatidylcholine in chloroform and wait for 5 min. Keep working under fume hood, remove the carriers, and transfer them in an empty crystallize and wait 5 min. Store them at room temperature in a closed box.
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2.
Heat an agarose low gelling 4% glass tube with a Bunsen burner to melt agarose. Keep it in a heating block at 55 °C. Transfer a glass tube containing water in the heating block. After putting the adults in the 200 μm deep flat carriers, preheat a Pasteur pipette by pipetting up and down 55 °C water. Pipet, immediately, a few microliters of agarose low gelling and quickly pour a small drop on the 200 μm deep flat carriers containing the adults.
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3.
During the whole time of manipulation, keep working in the cold compartment of the AFS2, with the minimal lighting in the room. Remember to put a cache on the porthole when you do not manipulate in the AFS2. Check regularly the level of liquid nitrogen in the AFS.
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4.
To limit photobleaching of GFP, set the light of the ultramicrotome and the room at minimum level.
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5.
This process is generally called trimming and is essential to prepare the sample for optimal thin sectioning.
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6.
It is not necessary to contrast grid with uranyl acetate and lead citrate because there is enough contrast with freeze substitution medium. Additionally, the section integrity is better conserved.
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7.
Add enough water to avoid the grid to be compressed, but not too much to avoid the grid to move during the observation.
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8.
Thin sections on the grids are fragile, therefore the fluorescence observation should be performed for a very short duration.
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9.
Centre the grid as much as possible; otherwise the correct focussing of the image could be difficult.
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10.
To superimpose the area of interest manually on the composite image, use different landmarks like the outline of the worm or the cuticle, small holes, or dirt.
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11.
A free open-source software called eC-CLEM, which could facilitate the correlation process, has been recently proposed [15]. It is available as a plugin in the Icy platform.
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23 January 2020
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Acknowledgments
The authors would like to thank the C. elegans community and the Caenorhabditis Genetic Center which is funded by the NIH National Center for Research Resources (NCRR) for sharing C. elegans strains. We are grateful to Christophe Lefebvre for Fig. 2 and Claire Boulogne for critically reading the manuscript. The present work has benefited from the expertise of Béatrice Satiat-Jeunemaitre, Cynthia Gillet, Jessica Marion, and Claire Boulogne and the core facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr) member of IBiSA (http://www.ibisa.net), supported by “France-BioImaging” (ANR-10-INBS-04-01) and the Labex “Saclay Plant Science” (ANR-11-IDEX-0003-02). This work was supported by the Agence Nationale de la Recherche (project EAT, ANR-12-BSV2-018) and the Association pour la Recherche contre le Cancer (SFI20111203826).
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Largeau, C., Culetto, E., Legouis, R. (2019). Subcellular Localization of ESCRT-II in the Nematode C. elegans by Correlative Light Electron Microscopy. In: Culetto, E., Legouis, R. (eds) The ESCRT Complexes. Methods in Molecular Biology, vol 1998. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9492-2_4
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