Dec 26, 2023

Public workspaceA polarized cell system amenable to subcellular resolution imaging of influenza virus infection

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.36wgq3pmklk5/v1
  • Brault Jean-Baptiste1,
  • Thouvenot Catherine2,3,
  • Cannata Serio Magda1,
  • Paisant Sylvain1,
  • Fernandes Julien4,
  • Gény David5,
  • Danglot Lydia6,7,
  • Mallet Adeline2,3,
  • Naffakh Nadia1
  • 1Institut Pasteur, Université Paris Cité, CNRS UMR 3569, RNA Biology of Influenza Viruses, 75015 Paris, France;
  • 2Institut Pasteur, Université Paris Cité, Ultrastructural BioImaging Unit, 75015 Paris, France;
  • 3Institut Pasteur, Université Paris Cité, Photonic BioImaging Unit, 75015 Paris, France;
  • 4Institut Pasteur, Université Paris Cité, C2RT, Unit of Technology and Services Photonic BioImaging, 75015 Paris, France;
  • 5Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, NeurImag facility, Université Paris Cité, 75014 Paris, France;
  • 6Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, NeurImag facility, Université Paris Cité, 102 rue de la santé, 75014, Paris, France;
  • 7Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Membrane Traffic in Healthy and Diseased Brain team, Université Paris Cité, 75014 Paris, France
  • Brault Jean-Baptiste: corresponding author;
  • Cannata Serio Magda: Current address: Institut Curie, PSL Research University, CNRS UMR144, 75005 Paris, France;
  • Naffakh Nadia: corresponding author;
  • PLOS ONE Lab Protocols
    Tech. support email: plosone@plos.org
Nadia Naffakh
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DOI: dx.doi.org/10.17504/protocols.io.36wgq3pmklk5/v1
External link: https://doi.org/10.1371/journal.pone.0292977
Protocol Citation: Brault Jean-Baptiste, Thouvenot Catherine, Cannata Serio Magda, Paisant Sylvain, Fernandes Julien, Gény David, Danglot Lydia, Mallet Adeline, Naffakh Nadia 2023. A polarized cell system amenable to subcellular resolution imaging of influenza virus infection. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3pmklk5/v1
Manuscript citation:
Brault J, Thouvenot C, Serio MC, Paisant S, Fernandes J, Gény D, Danglot L, Mallet A, Naffakh N (2024) A polarized cell system amenable to subcellular resolution imaging of influenza virus infection. PLOS ONE 19(1). doi: 10.1371/journal.pone.0292977
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it's working
Created: November 28, 2023
Last Modified: December 26, 2023
Protocol Integer ID: 92442
Funders Acknowledgements:
Human Frontiers Science Program
Grant ID: HFSP-RGP0040/2019
Agence Nationale de la Recherche
Grant ID: ANR-21-CE11-0010-03
Agence Nationale de la Recherche
Grant ID: ANR-10-INSB-04-01
Agence Nationale de la Recherche
Grant ID: ANR-10-INSB-10
Agence Nationale de la Recherche
Grant ID: ANR-10-LABX-62-IBEID
Abstract
This protocol details a polarized cell system amenable to subcellular resolution imaging of influenza virus infection.
Attachments
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A polarized cell sys...
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Materials
Plastic consumables


ABC
1 mL micropipettes Eppendorf861172
1.5 mL Eppendorf tubeEppendorf0030125150
100mm diameter culture dishCorning353003
15 mL tubeFalcon352097
6-well plateFalcon353047
12-well plateFalcon353043
24-well plateFalcon353046
Rubber padApplied BiosystemsN8010550

Cell culture reagents:
ABC
PBS (CaCl2-)Gibco14190
DMEM 1X (+) 4.5g/L D-Glucose L-Glutamine (-) pyruvateGibco41965
Fetal Bovine Serum (FBS) qualifiedGibco10437
Penicillin streptomycinGibco15140
Trypsin-EDTA 1X 0.05%Gibco25300
GelriteSigmaG1910
Cytodex 3 microcarrier beadsSigmaC3275
Cell culture medium

D10 medium: DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/ml streptomycin

Reagents for electron microscopy
ABC
Paraformaldehyde solutionEuromedex15714
Glutaraldehyde solution Grade ISigmaG5882-10
Osmium tetroxideEuromedex3000154760
Uranyl acetateElectron Microscopy Science22400
Ethanol absoluteFisher scientific10680993
Epoxy resinLFG14901
Lead citrateDelta microscopies11300
HEPESThermoFisher Scientific15630080
DMP30LFG13600
Tannic acidSigma1401-55-4
Potassium ferricyanideSigma14459-95-1
EGTAFisher Scientific11514736
PIPESSigmaP6757
PBSFisher scientific12559069
Toluidine blueFluka AG89640
PHEM Buffer Delta microscopiesGT-140174
Epoxy resin solution for electron microscopy:

  • Mix Epon (166 mL), DDSA (100 mL) and NMA (84 mL) slowly for 20 min, avoid bubbles.
  • Add 170 µl of DMP30 to 10 mL of the Epoxy resin solution extemporaneously before embedding.

Equipment for electron microscopy:
ABC
Fine forceps Dumont, Style 7EMS72801-D
Electronic balanceMettler toledoME2002
Razor blade (single Edge Carbon Steel)EMS71960
IncubatorMemmertN/A
ultramicrotomeLeicaUC7
Formvar carboned grids 200 MeshEMSFCF200-Cu-50
Diamon knife Histo 45° 8mmLFG - DiatomeN/A
Diamon knife Ultra 45° 3mmLFG - DiatomeN/A
Glass slideFisher scientific10090431
Transmission electron microscopeThermoFisherTechnai Biotwin T12
StereomicroscopeLeicaN/A
HotplateSartoriusN/A
Ashless quantitative filter paper (55mm)Whatman                                  140055
Reagents for confocal microscopy:

ABC
Paraformaldehyde 32% w/vThermo scientific47377
NH4ClSigma213330
Tween 20SigmaP1379
Triton X100SigmaT9284
Antifade mounting mediumVectashieldH-1000
DAPIThermoScientific62248
Normal donkey serumMerckS30
Normal goat serumMerckS26
Gelatin from cold water fish skinSigmaG7765
Buffers for confocal microscopy:

  • Blocking buffer: 5% donkey serum, 3% goat serum, 0.25% fish skin gelatin, 0.2% Triton X100
  • Immuno-staining buffer: 5% donkey serum, 3% goat serum, 0.125% fish skin gelatin, 0.2% Triton X100

Primary Antibodies for confocal microscopy:

ABC
Mouse monoclonal anti Influenza A NP clone AA5H BIO-RADMCA400 (1:500)
Rabbit polyclonal anti MPP5/PALS-1 Proteintech17710-1-AP (1:500)
Mouse monoclonal anti ZO-1, clone 1A12Invitrogen33-9100 (1:100)
Rabbit polyclonal anti RAB11AInvitrogen71-5300 (1:100)
Secondary Antibodies for confocal microscopy:
ABC
Goat anti mouse IgG Alexa Fluor555 Invitrogen A-21424
Donkey anti rabbit IgG Alexa Fluor488InvitrogenA-21206
Donkey anti-rabbit IgG Alexa Fluor594InvitrogenA-21207
Goat anti-mouse IgG Atto 647NMerck50185
Equipment for confocal microscopy:
ABC
Cell strainer 70µm Nylon Corning 431751
Secure-seal spacer one wellInvitrogenS24735
Slides Superfrost Plus AdhesionEprediaJ1800AMNZ
Coverslips Epredia CB00130RAC20MNZ0
Wheel Fisherbrand88861050
TCS SP8 scanning confocal microscopeLeicaN/A

Softwares and algorithms:
ABC
TIAThermoFisherN/A
Image JNIHN/A
LasXLeicaN/A
ImarisOxford InstrumentsN/A
ReagentEppendorf Tubes® 3810XEppendorfCatalog #0030125150
ReagentFalcon® 100 mm TC-treated Cell Culture Dish, 20/Pack, 200/Case, SterileCorningCatalog #353003
ReagentFalcon 15mL Conical Centrifuge TubesCorningCatalog #352097
ReagentFalcon® 24-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Sterile, 50/CasCorningCatalog #353047
ReagentFalcon® 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Individually WrCorningCatalog #353043
ReagentFalcon® 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Individually WraCorningCatalog #353046
Reagent1x DPBSGibco - Thermo FischerCatalog #14190144
ReagentFetal Bovine SerumGibco - Thermo FisherCatalog #10437028
ReagentPenicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
Reagent0.05% Trypsin-EDTA (1X)Thermo Fisher ScientificCatalog #25300-054
ReagentGelzan™ CMMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1910
ReagentCytodex® 3 microcarrier beadsMerck MilliporeSigma (Sigma-Aldrich)Catalog #C3275
ReagentParaformaldehyde 32% (methanol free)Electron Microscopy SciencesCatalog #15714
ReagentGlutaraldehyde solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5882
ReagentEthanol 99%+, Absolute, Extra Pure, SLR, Fisher Chemical™Fisher ScientificCatalog #10680993
Reagent Lead Citrate (Reynolds)Delta microscopiesCatalog #11300
ReagentHEPES 1MThermo Fisher ScientificCatalog #15630080
ReagentTannic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #1401-55-4
Reagent Potassium hexacyanoferrate(II) trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog # 14459-95-1
ReagentInvitrogen™ EGTA, Tetra(acetoxymethyl Ester) (EGTA, AM)Fisher ScientificCatalog #11514736
ReagentPIPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6757
ReagentGibco™ DPBS, no calcium, no magnesiumFisher ScientificCatalog #12559069
ReagentParaformaldehyde, 32% w/v aq. soln., methanol free, Thermo Scientific ChemicalsThermo Fisher ScientificCatalog #047377.9L
ReagentAmmonium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #213330
ReagentTween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T9284
ReagentVECTASHIELD Mounting MediumVector LaboratoriesCatalog #H-1000
ReagentDAPIThermo Fisher ScientificCatalog #62248
ReagentDonkey Serum, 100 mlMerck MilliporeSigma (Sigma-Aldrich)Catalog #S30-M
ReagentGoat Serum, 100 mlMerck MilliporeSigma (Sigma-Aldrich)Catalog #S26-100ML
ReagentGelatin from cold water fish skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7765
ReagentInfluenza A Nucleoprotein antibody | AA5HBio-Rad LaboratoriesCatalog #MCA400
ReagentMPP5 Polyclonal antibodyProteintechCatalog #17710-1-AP
ReagentZO-1 Monoclonal Antibody (ZO1-1A12)Thermo Fisher ScientificCatalog #33-9100
ReagentRAB11A Polyclonal AntibodyThermo Fisher ScientificCatalog #71-5300
ReagentGoat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555Thermo Fisher ScientificCatalog #A-21424
ReagentDonkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Thermo Fisher ScientificCatalog #A-21206
ReagentDonkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594Thermo Fisher ScientificCatalog #A-21207
ReagentAnti-Mouse-IgG - Atto 647N antibody produced in goatMerck MilliporeSigma (Sigma-Aldrich)Catalog #50185
ReagentCorning® cell strainerCorningCatalog #CLS431751-50EA
ReagentSecure-Seal™ Spacer, one well, 13 mm diameter, 0.12 mm deepThermo FisherCatalog #S24735

SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
SEEDING AND POLARIZATION OF CACO-2/TC7 CELLS ON CYTODEX 3 BEADS
15m
15m
Prepare the repellent layer and seed the Caco-2/TC7 cells
Dissolve the Gelrite solution (0.8% Gelrite + 0.1% MgSO4 x7H2O w/v in PBS) in the microwave oven.
Distribute the Gelrite solution (Amount2.5 mL /well) in a 35 mm dish or a well of a 6-well plate. Let it solidify and cool down at TemperatureRoom temperature for about Duration00:10:00 .

10m
Wash a 100 mm diameter culture dish of subconfluent Caco-2/TC7 cells with Amount5 mL of PBS.

Wash
Add Amount1 mL of trypsin and incubate Duration00:05:00 at Temperature37 °C .

5m
Pipetting
Add Amount9 mL of DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin (D10 medium). Pipet up and down to homogenize the cell suspension.

Pipetting
Count the cells. Prepare Amount2 mL of a cell suspension at 2,25 x 105 cells/mL.

Add Amount50 µL of the Cytodex 3 suspension (~1500 beads/50 µL) to the cell suspension, mix gently and add to the Gelrite layer. Place into the cell incubator onto a rubber pad to attenuate the incubator’s vibrations.

Pipetting
Mix
The next day, homogenize the beads suspension by gently pipetting up and down 5x with a 1000 µL pipetman tip, place them back in the cell incubator.
After three days, filter out the non-attached cells
Place a 70 µm cell strainer over a 50 mL tube.
Collect the beads suspension and pass it through the cell strainer. Discard the flow-through.
Flip the filter with sterile tweezers over the Gelrite layer, add Amount2 mL of fresh D10 medium on the top of the flipped filter to recover the beads.

Pipetting
Change the medium twice a week by removing the medium carefully while leaving the beads at the surface of the Gelrite layer, and replacing it with fresh D10 medium. Keep going for 10-15 days.
INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
INFLUENZA A VIRUS INFECTION (in a BSL2+ laboratory)
1h
1h
Transfer the Cytodex beads from the Gelrite layer to a 15mL tube. Let them sediment at the bottom of the tube.
Remove gently the medium, add Amount1 mL of prewarmed (Temperature37 °C ) DMEM for washing, and let the beads sediment at the bottom of the tube.

Pipetting
Repeat the washing step once, then transfer the beads to a 1.5mL tube.
Let the beads sediment and remove gently the medium, resuspend the beads in Amount250 µL of a suspension of Influenza A virus (A/WSN/1933(H1N1)) at a high titer. Given the estimated numbers of ~ 1500 beads/sample and ~400 cells/bead, a ~2,5 x107 PFU/mL titre will lead to an MOI of ~10 PFU/cell.

Incubate the beads at Temperature37 °C for Duration01:00:00 , and gently tap the tube with fingertip every 15 mn to resuspend the beads.

1h
Incubation
Let the beads sediment at the bottom of the tube, remove the viral inoculum, add Amount1 mL of PBS for washing and let the beads sediment at the bottom of the tube.

Pipetting
Remove the PBS, add Amount1 mL of DMEM supplemented with 2% FBS, resuspend the beads and place them in a well of a 12-well culture plate. Place the plate in a cell incubator at Temperature37 °C for the selected period of time (typically, 2-6 hours to investigate early stages of the viral life cycle, 8-24 hours to investigate late stages).

Pipetting
CONFOCAL IMAGING
CONFOCAL IMAGING
6h
6h
Fix the cells
Remove the medium carefully while leaving the beads at the bottom of the well. Resuspend the beads in 250µL of PBS-4% PFA. Incubate at room temperature for Duration00:20:00 .

20m
Transfer the beads suspension in a 1.5mL tube. Let the beads sediment at the bottom of the tube. Remove gently the PBS-4% PFA, add 1mL of PBS for washing, and let the beads sediment at the bottom of the tube.
Repeat the washing step twice, then transfer the beads to a 1.5mL tube.
Note
At this stage, the sample can be considered no-longer infectious and can therefore be manipulated outside the BSL2+ facility.
Let the beads sediment at the bottom of the tube. Remove the PBS and add 1mL of PBS-50mM NH4Cl for 10 minutes at room temperature, to quench the residual PFA.
Wash the beads 2x with 1mL PBS as described above.
Immunostain the cells
Let the beads sediment at the bottom of the tube. Remove the PBS and add Amount500 µL of the blocking buffer. Place the tube on a spinning wheel at Temperature4 °C for Duration01:00:00 , with a rotation speed of Centrifigation15 rpm .

1h
Centrifigation
Pipetting
Let the beads sediment at the bottom of the tube. Remove the blocking buffer and add Amount250 µL of the primary antibody diluted in the immuno-staining buffer. Place the tube on a spinning wheel at Temperature4 °C DurationOvernight , with a rotation speed of Centrifigation15 rpm .

1h
Pipetting
Overnight
The next day, wash the beads 3x with Amount1 mL of PBS-0.05% Tween 20 as described above.

Wash
Let the beads sediment at the bottom of the tube. Remove the PBS-0.05% Tween 20 and add Amount250 µL of the secondary antibody diluted in the immunostaining buffer. Place the tube on a spinning wheel at Temperature4 °C for Duration02:00:00 , with a rotation speed of Centrifigation15 rpm .

2h
Centrifigation
Pipetting
Wash the beads 2x with Amount1 mL of PBS- 0.05% Tween 20 as described above.

Wash
Wash the beads 2x with Amount1 mL of PBS as described above.

Wash
Wash the beads 2x with Amount1 mL of distilled water as described above.

Wash
Let the beads sediment at the bottom of the tube and remove the water. Use a very thin 10µL pipetman tip to remove residual water and to dry out the beads pellet. Immediately add Amount50 µL of Vectashield mounting medium over the beads pellet, avoiding the formation of bubbles.

Pipetting
Gently tap the tube with fingertip to resuspend the beads.
Place an adhesive secure-seal spacer on a clean glass microscope slide.
Cut the extremity of 200µL pipetman tip to widen it, gently pipet out the beads resuspended in Vectashield mounting medium, avoiding the formation of bubbles. Drop them at the center of the spacer on the glass microscope slide.

Cover the drop with a sterile #1.5 round glass microscope coverslip. Protect from light and let at TemperatureRoom temperature DurationOvernight and seal with nail polish.

2h
Overnight
Acquire confocal images.
Select the 40x NA 1,3 oil immersion objective and apply appropriate oil.
Install sample on the slide holder of the microscope.
Create the lightpath configuration corresponding to the staining used. Create three imaging sequence:
  1. DAPI : Excitation 405 nm, Emission : 416-501 nm
  2. PALS-1 : Excitation 598 nm, Emission : 608-643 nm
  3. NP : Excitation 646 nm, Emission : 656-705 nm
Find your sample, adjust focus.
Laser power and gain settings will be set according to the intensity of the labelling in order not to saturate the signal.
Define your Z-stack by selecting the top and the bottom of your sample. Define Z step size to 0,35 µm.
Set acquisition parameters: Set frame size to 1024x1024 pixels (0,3 µm/pixel), scan speed to 400 Hz in a bi-directional mode.
Start image acquisition.
Acquire confocal images at high magnification.
Select the HC PL APO 93x NA 1,3 glycerol immersion objective and apply appropriate glycerol.
Install sample on the slide holder of the microscope.
Adapt the motorized objective colar to match the sample index, using reflection mode in XZY mode.
Create the lightpath configuration corresponding to the staining used. Create three imaging sequence:
  1. Atto 647N : Excitation 670 nm, Emission : 654-719 nm
  2. AlexaFluor 594 : Excitation 594 nm, Emission : 604-641 nm
  3. Stargreen : Excitation 470 nm, Emission : 505-561 nm
Find your sample, adjust focus.
Laser power and gain settings will be set according to the intensity of the labelling in order not to saturate the signal. HyD detectors are used to favor sensitivity.
Define your Z-stack by selecting the top and the bottom of your sample. Define Z step size to 0,18 µm.
Set acquisition parameters: scan speed to 400 Hz in a bi-directional mode, Frame Average2. Adapt zoom, matrix size and Z step to be in ideal sampling (here pixel size 85 nm).
ELECTRON MICROSCOPY IMAGING
ELECTRON MICROSCOPY IMAGING
1d 12h 5m 20s
1d 12h 5m 20s
Fix the cells.
Remove the medium carefully while leaving the beads at the bottom of the well. Resuspend the beads in Amount250 µL of PBS-3% PFA-0.1% glutaraldehyde. Leave at Temperature4 °C DurationOvernight .

2h
Overnight
Transfer the beads suspension in a 1.5mL tube. Let the beads sediment at the bottom of the tube. Remove gently the PBS-3% PFA-0.1% glutaraldehyde, add Amount250 µL of PHEM-2.5% glutaraldehyde. Leave at Temperature4 °C for Duration24:00:00 .

Note
At this stage, the sample can be considered no-longer infectious and can therefore be manipulated outside the BSL2+ facility.



1d
Let the beads sediment at the bottom of the tube. Remove gently the PHEM-2.5% glutaraldehyde, add Amount1 mL of PHEM, incubate for Duration00:05:00 at TemperatureRoom temperature .
5m
Incubation
Pipetting
Repeat the PHEM washing 2x.
Perform uranyl acetate staining.
Let the beads sediment at the bottom of the tube. Remove gently the PHEM, add Amount250 µL of the PHEM-1% Osmium tetroxide-1.5% potassium ferrocyanide post-fixation buffer. Incubate for Duration01:00:00 at TemperatureRoom temperature .

1h
Incubation
Pipetting
Wash 3x in PHEM as described above.
Wash
Wash 3x in filtered distilled water, with 5 mn incubation times as described above.
Wash
Let the beads sediment at the bottom of the tube. Remove gently the water, add Amount250 µL of water supplemented with 0.2% tannic acid. Incubate Duration00:30:00 at TemperatureRoom temperature .

30m
Incubation
Pipetting
Wash 3x in filtered distilled water, with Duration00:05:00 incubation times as described above.

5m
Incubation
Wash
Let the beads sediment at the bottom of the tube. Remove gently the water, add Amount250 µL of water supplemented with 2% osmium. Incubate Duration00:30:00 at TemperatureRoom temperature .

30m
Incubation
Pipetting
Wash 3x in filtered distilled water, with Duration00:05:00 incubation times as described above.

5m
Wash
Let the beads sediment at the bottom of the tube. Remove gently the water, add Amount250 µL of 25% ethanol in water (v/v)- 1% Uranyl Acetate. Incubate Duration01:00:00 at TemperatureRoom temperature (protect from light).

1h
Incubation
Pipetting
Embed the samples.
Let the beads sediment at the bottom of the tube. Remove gently the uranyl acetate solution. Add Amount1 mL of 50% Ethanol in water (v/v). Incubate Duration00:05:00 at TemperatureRoom temperature .

5m
Incubation
Pipetting
Repeat step 16.8 with 75% ethanol in water (v/v).
Repeat step 16.8 with 100 % ethanol.
Prepare the Epoxy resin at TemperatureRoom temperature .(https://www.emsdiasum.com/docs/technical/datasheet/14120).

Let the beads sediment at the bottom of the tube and remove gently the ethanol with a micropipette.
Add Amount200 µL of Epoxy resin onto the beads and homogenize slowly with back-and-forth movements with the micropipette.

Pipetting
Let the beads sediment at the bottom of the tube. Remove Amount150 µL of Epoxy resin and add Amount150 µL of fresh Epoxy resin.

Pipetting
Wait Duration03:00:00 of impregnation time with the tubes open under the chemical hood.

3h
Incubate DurationOvernight at TemperatureRoom temperature to allow polymerization of the resin.

3h
Incubation
Section the samples with an ultramicrotome.
Use a fresh blade to trim away excess resin from the block underneath the embedded compound eye under the stereomicroscope. Shape the upper part into an equilateral trapezoid, and shape the lower part into a square base.
Place the shaped block into the chuck on the ultramicrotome and tighten it firmly.
Place the HISTO diamond sectioning knife on the sectioning knife holder stage on the ultramicrotome.
Add a little excess water to the groove of the diamond sectioning knife to make it convex and stop when the full length of the diamond blade is soaked.
Pipetting
Gently draw a portion of the water under the microscope with a syringe or Pasteur pipette. Adjust the lightning system so that when the liquid surface becomes concave, a curved surface reflecting light can be seen, which is the correct liquid surface and appears white under the microscope.
Note
As the liquid evaporates during sectioning, distilled water should be added to ensure the correct liquid level.

Pipetting
Imaging
Adjust the knife angle so that the two parallel sides of the trapezoid sample are parallel to the blade.
Move the knife to align the available part with the block surface.
Raise the block until it stops slightly above the knife blade.
Set parameters in the control panel: feed of 1000 nm, speed of 1 mm/s, thickness 1 µm. Start the automated movement of the ultramicrotome specimen arm.
Cut semi-thin sections (Thikness1 µm thick). The sections float on the water in the bowl of the HISTO diamond knife.

Collect the sections in a drop of water with a handle and place them on a glass slide.
Stain the semi-thin sections by adding a drop of Amount50 µL of toluidine blue (5% p/v) on the sections and incubating the samples 2 mn on a hot plate (Temperature60 °C ).

Pipetting
Look at the stained semi-thin sections on the glass slide with a photonic microscope. Repeat the steps 18.4 to 18.9 until an area of interest with beads is observed.
Halt sectioning and replace the HISTO diamond knife with an Ultra 45° diamond knife. Fill the knife reservoir with fresh distilled water. Adjust the liquid level and sectioning parameters: feed of 70 nm, speed of 1 mm/s.
Cut ultrathin sections (70 nm thick). The sections float on the water in the bowl of the Ultra 45° diamond knife.
Carefully collect 2 sections on a carboned grids.
Place the grids with thin sections into the transmission electron microscope sample box.
Staining of ultrathin sections.
Prepare a parafilm of 15 cm in width and place it on a delimited radioactive area, with the clean surface of the parafilm on the upper side.
For each grid, add one drop (about Amount100 µL ) of water supplementmented with 4% Uranyl Acetate, filtered (0.22µm), on the parafilm.

Pipetting
Place gently the grid (section side) on the droplet surface and let it stand for Duration00:40:00 . Protect it from light.

40m
For each grid, add three drops (about Amount200 µL ) of distilled water on a clean area of the parafilm. Place the grid on the first of the three droplets before moving it on the second and then on the third droplet. Wait Duration00:00:10 before transferring a grid to the next droplet.

10s
For each grid, add one drop of water supplemented with 1% lead citrate, filtered (0.22 µm), on a clean area of the parafilm.
Pipetting
Place the grid on the top of the droplet and let it stand for Duration00:05:00 .

5m
For each grid, add five drops of fresh distilled water on a clean area of the parafilm. Wash the grids by placing them successively on the five droplets, as in step 19.4. Wait Duration00:00:20 before transferring a grid to the next droplet.

20s
Pipetting
Wash
After drying the grids on a filter paper, place them in the transmission electron microscope sample box.
Electron microscopy imaging.
Acquire images with a transmission electron microscope at 120 kV.
Collect images of cells on beads.