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Tubulin, Atto-565 labelled tubulin, and biotinylated tubulin are prepared as already fully described in Ramirez-Rios et al. (2017). Store up to 1 year in liquid nitrogen
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Cover glasses 26 × 76 mm #1 (VWR, catalog number: 630-2910)
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Double-face precut tape 70 µm thick, 3 mm wide (LIMA Company, catalog number: 0000P70PC3003)
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Siligum wax plate (VWR, MODU140013)
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1.5 ml Eppendorf tubes (Fisher Scientific, catalog number: 11558232)
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0.5 ml Eppendorf tubes (Fisher Scientific, catalog number: 10318661)
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Petri dishes (Greiner Bio-One, catalog number: 663102)
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Polycarbonate centrifuge tubes (Beckman, catalog number: 343775)
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0.22 µm filters (Merck Millipore, catalog number: SLGP033RS)
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Silane-PEG (MW 30k) (Creative PEG-Works, catalog number: PSB-2014)
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Silane-PEG-biotin (MW 3,400) (LaysanBio, catalog number: BIOTIN-PEF-SIL, MW 3,400)
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NeutrAvidin (ThermoScientific, catalog number: 31000)
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PLL20K-G35-PEG2K (PLL-g-PEG) (Jenkem, catalog number: 13022755)
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Pluronic F-127 (Sigma-Aldrich, catalog number: P2443)
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Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7030)
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Acetone 100% (VWR, catalog number: 20066.321)
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Ethanol 96% (VWR, catalog number: 20823362)
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Hellmanex III (Sigma-Aldrich, catalog number: Z805939-1EA)
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Phosphate buffer saline (PBS) (Sigma-Aldrich, catalog number: P4417)
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1,4-Piperazinediethanesulfonic acid (PIPES) (Sigma-Aldrich, catalog number: P6757)
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Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 484016)
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Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541)
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Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889)
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Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: 2670)
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Sodium hydroxide (NaOH) (Carlo Erba, catalog number: 480507)
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DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)
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Guanosine 5′-triphosphate (GTP) (Sigma-Aldrich, catalog number: G8877)
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Methyl cellulose 1,500 cP (Sigma-Aldrich, catalog number: M0387)
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GMPCPP (Euromedex, JE-NU-405S)
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HEPES (Sigma-Aldrich, catalog number: H3375)
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Glucose (Sigma-Aldrich, catalog number: G8270)
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Glucose oxidase (Sigma-Aldrich, catalog number: G6766-10KU)
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Catalase (Sigma-Aldrich, catalog number: C9322-1G)
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Diamond pencil (Agar Scientific, catalog number: AGT5347)
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Glutaraldehyde solution 25% (Sigma-Aldrich, catalog number: G5882)
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Clean nitrogen air flow (Air Liquide)
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Liquid nitrogen (Air Liquide)
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5× BRB80 (see Recipes)
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BSA 10% (see Recipes)
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Neutravidine (see Recipes)
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DTT, 200 mM (see Recipes)
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KCl, 500 mM (see Recipes)
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PLL-PEG (see Recipes)
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Silane-PEG or Silane-PEG-biotin (see Recipes)
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GTP, 20 mM (see Recipes)
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Glucose, 450 mg/ml (see Recipes)
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Deoxymix (catalase and glucose oxydase) (see Recipes)
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NaOH, 1 M (see Recipes)
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BSA 1%/BRB80 (see Recipes)
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Methyl cellulose 1,500 cP (see Recipes)
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HEPES, 10 mM (see Recipes)
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Pluronic F27, 10% (see Recipes)
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PBS (see Recipes)
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Neutravidin stock solution (see Recipes)
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Red tubulin mix (see Recipes)
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Imaging buffer (see Recipes)
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Inverted Eclipse Ti microscope (Nikon) with PSF focus
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Apochromat 100×/1.49 N.A. oil immersion objective (Nikon) with heated objective (Okolab)
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Temperature chamber (Technico Plast)
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Ilas2 TIRF system (Roper Scientific)
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Cooled Charged-coupled device camera (Evolve 512, Photometrics)
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Temperature Stage Controller (Linkam Scientific)
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LSM 710 confocal (Zeiss) Airyscan
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Plan Apochromat 100×/1.4 N.A. oil immersion objective (Zeiss)
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Ultracentrifuge (Beckman Coulter Optima, Model Max-XP, catalog number: 393315)
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Rotor (Beckman, model: TLA100-1, catalog number: 343837)
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Plasma cleaner FEMTO Diener Electric (Germany) coupled to a vacuum pump Trivac D2.5E Oerliken (Germany)
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Sonicator (Elmasonic S30, Elma, Germany)
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Glass staining dishes and tray (VWR, catalog number: MARI4200004)
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Plastic box for glass microscopy (any brand)
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Diamond Pencil (Oxford Instruments, catalog number: T5347)
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Tweezers (Dutscher, catalog numbers: 076100 and 005093)
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Gloves powder-free (any brand)
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Water-bath (any brand)
All solutions were conserved at indicated temperature without any observable deterioration along time unless stated otherwise. Most solutions were aliquoted to avoid freezing-thawing cycles.
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Neutravidine
Dissolve 10 mg Neutravidine in 10 ml H2O, aliquot (5 and 100 µl) and store at -20 °C
Before the experiment, predilute 5 µl with 195 µl of 1% BSA/BRB80 (final concentration 25 µg/ml)
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DTT, 200 mM
Dissolve 617 mg DTT powder in 4 ml deionized H2O, filtrate, aliquot and store at -20 °C
The day of use, dilute 1/5 in H2O or BRB80. Discard aliquot after each day of use
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KCl, 500 mM
Dissolve 1 g KCl into 50 ml deionized H2O (-20 °C) filtrate, aliquot and store at -20 °C
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PLL-PEG
-
Make aliquot of the powder of known weight (20-30 mg) under argon gas and store at -20 °C.
-
To make stock solution, dissolve the powder in 10 mM HEPES (pH 7.4) at 1 mg/ml, make 50 µl aliquots and store at -20 °C.
-
The day of use, dilute one 50 µl aliquot with 450 µl of 10 mM HEPES (pH 7.4). Diluted PLL-PEG can be stored at 4 °C for one week.
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Silane-PEG or Silane-PEG-biotin
Notes:
-
Silane-PEG or Silane-PEG-biotin powders are weighted and aliquoted in 200 mg per eppendorf tube under argon gas. The tubes are tightly sealed with parafilm and stored at -20 °C.
-
Silane-PEG and Silane-PEG-biotin solutions must be kept anhydrous and in the dark.
-
Dissolve 200 mg powder in 200 ml of 96% ethanol plus 0.4 ml of 37% HCl.
-
To solubilize the Silane-PEG solution, heat to 50 °C in a water-bath. Keep the solution in a glass bottle.
-
Silane-PEG and silane-PEG-biotin solution are stored at room temperature in the dark for up to 4 months or around 15 coating. If experiments look dirty in the background, use freshly prepared coating solutions.
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GTP 20 mM
Dissolve 250 mg GTP in 5 ml H2O
Make aliquots and store at -20 °C
Before use, dilute the stock solution to 20 mM in H2O or BRB80
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Glucose 450 mg/ml
Dissolve 2.250 g of glucose in 5 ml of BRB80
Filtrate, aliquot and store at -80 °C
The day of use pre-dilute 1/10 in BRB80 and keep on ice
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Deoxymix (catalase and glucose oxydase)
Dissolve 35 mg of catalase plus 250 mg of glucose-oxydase in 10 ml of BRB80
Filtrate, make 25 µl aliquots, freeze in liquid nitrogen and store at -80 °C
Discard thawed aliquot after each day of use
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NaOH, 1 M
Dissolve 8 g of NaOH in 200 ml deionized water
Discard after each use
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BSA 1%/BRB80
Dilute 500 µl of 10% BSA with 1 ml of BRB80 5× in 3.5 ml of filtrated deionized water
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Methyl cellulose 1,500 cP
Dissolve 100 mg of methyl cellulose 1,500 cP in 10 ml of prewarm (60 °C) deionized water
Gently shake on rotating wheel for 30 min
Store at 4 °C for two weeks
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HEPES, 10 mM
Dissolve 477 mg in 200 ml deionized water
Adjust pH to 7.4 with KOH
Store in aliquots at -20 °C
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Pluronic F27, 10%
Dissolve 100 mg in 1 ml of deionized water
Store at 4 °C up to 2 months
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PBS
Dissolve 1 tablet in 200 ml of deionized water
Store at -20 °C
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Neutravidin stock solution
Dissolve Neutravidin in deionized water at a final concentration of 1 mg/ml
Store at -20 °C
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Red tubulin mix
Dilute purified non-labelled and ATTO-565 tubulin at a 9:1 molar ratio in BRB80
Spin down aggregates at 100,000 × g, 4 °C using TLA100 rotor
Estimate tubulin concentration by measuring the OD280nm (1OD280nm = 1 mg/ml = 10 µM of tubulin)
Aliquot, quickly freeze and store in liquid nitrogen for up to 1 year
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Polymerization mix
BRB80 supplemented with:
50 mM KCl
1% BSA
4 mM DTT
1 mM GTP
1 mM glucose
0.05% Methyl cellulose 1,500 cP
1/50 deoxy mix (70 µg/ml catalase, 500 µg/ml glucose oxidase, 1 mg/ml glucose)
12 µM red tubulin mix
200 nM MAP6-N-GFP
Fundings: This work was supported by INSERM (Institut National de la Santé Et de la Recherche Médicale), CEA (Commissariat à l’Energie Atomique), CNRS (Centre National de la Recherche Scientifique), Université Grenoble Alpes, by grants from the Agence Nationale de la Recherche ANR MAMAs 2017-CE11-0026, ANR-15-IDEX-02 NeuroCoG in the framework of the “Investissements d’avenir” program and by fundings from the Ministère de l’enseignement supérieur et de la recherche. GIN is a member of the Grenoble Center of Excellence in Neurodegeneration (GREEN). The Photonic Imaging Center of Grenoble Institute Neuroscience (Univ. Grenoble Alpes–Inserm U1216) is part of the ISdV core facility and certified by the IBiSA label.
The authors declare no competing interests.