5. Glass capillaries with a filament (GD-1, Narishige or equivalent) are used to make injection needles with a glass capillary
puller (PC-10, Narishige or equivalent).
6. Aspirator tube assemblies for calibrated microcapillary pipettes
(Sigma-Aldrich, A5177-5EA or equivalent).
7. 1% Penicillin/Streptomycin (P/S) in MilliQ H 2 O.
8. Syringe, thin forceps, curved scissors and plastic tape.
2.2 Imaging Plate
Preparation
1. Millicell cell culture plate inserts (0.4 mm: Millipore,
PICMORG50).
2. 25 mL of thin albumen from eggs incubated overnight.
3. 10% glucose in MilliQ H 2 O.
4. 0.6% Granulated Agar (Difco, 0145-17-0) in MilliQ H 2 O.
5. 5 M NaCl in MilliQ H 2 O.
6. P/S, undiluted.
7. Water bath, 50 mL Falcon tubes and Pasteur pipettes.
2.3 Mounting and In
Vivo Time-Lapse
Imaging
1. Filter paper rings prepared from 2 Â 2 cm squares of Whatman
grade 1 filter paper in which a cloverleaf shaped hole is made in
the center with a paper punch, cutting the corners so that they
fit in the round imaging plates (Fig. 3a).
2. PBS 1Â (1 L): 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g
KH 2 PO 4 , 800 mL of MilliQ H 2 O, adjusted pH to 7.4 and to
1 L, sterilized by autoclaving and stored at room temperature.
3. 5 mL of thin albumen from eggs incubated overnight.
4. 5 mL of 123 mM NaCl in MilliQ H 2 O.
5. Soft tissues (Kimtech Science Kimwipes, or equivalent), thin
forceps, fine scissors, Pasteur pipettes, electrical insulation tape
and 100 mm petri dishes.
6. Culture chamber [14] created from a Corning
® Costar
® polystyrene 6-well plate (Sigma, CLS3736 or equivalent). To favor
the optics, the plastic in the lid is replaced with glass. In each
well, a 23 mm hole is made in the centre of the lid by pushing a
heated cork borer through the plastic, smoothing the rough
edges and sealing a 25-mm-diameter glass #1 coverslip over the
hole using Marine Adhesive (Zolux Silicone “SA 500”)
(Fig. 3d).
2.4 Image
Processing
and Analysis
1. ZEN software (Zeiss) Version 2.3 blue edition with the experiment designer option or any equivalent option allowing timelapse imaging with multiple XYZ positions.
2. Image J/Fiji software [39, 40].
3. BioFormat plugin [41] (https://imagej.net/Bio-Formats).
190
Elena Gonzalez-Gobartt et al.
puller (PC-10, Narishige or equivalent).
6. Aspirator tube assemblies for calibrated microcapillary pipettes
(Sigma-Aldrich, A5177-5EA or equivalent).
7. 1% Penicillin/Streptomycin (P/S) in MilliQ H 2 O.
8. Syringe, thin forceps, curved scissors and plastic tape.
2.2 Imaging Plate
Preparation
1. Millicell cell culture plate inserts (0.4 mm: Millipore,
PICMORG50).
2. 25 mL of thin albumen from eggs incubated overnight.
3. 10% glucose in MilliQ H 2 O.
4. 0.6% Granulated Agar (Difco, 0145-17-0) in MilliQ H 2 O.
5. 5 M NaCl in MilliQ H 2 O.
6. P/S, undiluted.
7. Water bath, 50 mL Falcon tubes and Pasteur pipettes.
2.3 Mounting and In
Vivo Time-Lapse
Imaging
1. Filter paper rings prepared from 2 Â 2 cm squares of Whatman
grade 1 filter paper in which a cloverleaf shaped hole is made in
the center with a paper punch, cutting the corners so that they
fit in the round imaging plates (Fig. 3a).
2. PBS 1Â (1 L): 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g
KH 2 PO 4 , 800 mL of MilliQ H 2 O, adjusted pH to 7.4 and to
1 L, sterilized by autoclaving and stored at room temperature.
3. 5 mL of thin albumen from eggs incubated overnight.
4. 5 mL of 123 mM NaCl in MilliQ H 2 O.
5. Soft tissues (Kimtech Science Kimwipes, or equivalent), thin
forceps, fine scissors, Pasteur pipettes, electrical insulation tape
and 100 mm petri dishes.
6. Culture chamber [14] created from a Corning
® Costar
® polystyrene 6-well plate (Sigma, CLS3736 or equivalent). To favor
the optics, the plastic in the lid is replaced with glass. In each
well, a 23 mm hole is made in the centre of the lid by pushing a
heated cork borer through the plastic, smoothing the rough
edges and sealing a 25-mm-diameter glass #1 coverslip over the
hole using Marine Adhesive (Zolux Silicone “SA 500”)
(Fig. 3d).
2.4 Image
Processing
and Analysis
1. ZEN software (Zeiss) Version 2.3 blue edition with the experiment designer option or any equivalent option allowing timelapse imaging with multiple XYZ positions.
2. Image J/Fiji software [39, 40].
3. BioFormat plugin [41] (https://imagej.net/Bio-Formats).
190
Elena Gonzalez-Gobartt et al.