Microinjections step by step.
This is a daily routine procedure of oocytes preparation for calcium measurements.
I. Making injection chambers.
Materials (picture):
- Coverslips 24x24 or 22x22 mm
- Diamond pen and ruler
- A couple of paper clips
- Forceps
- Paraffin plate in a Petri dish
- Spirit-lamp and lighter
Procedure:
1. Turn on the spirit-lamp.
2. Cut coverslip to have a 12x6 mm peace.
3. Insert one entire coverslip in a paper clip.
4. Put the small peace of coverslip in order to creat an angle about 200
5. Put a tiny peace of paraffin in the site of connection of two peaces of glass.
6. Pass the coversip above the fire to melt the paraffin.
7. Remove the clip after solidification of paraffin.
Eventually you should have the chamber shown in the
picture.
II. Making acquisition chambers.
Materials (picture):
- Coverslips 24x24 mm
- Diamond nife and ruler
- Double-face sckotch 60 um thick.
- Forceps
- Scissors
- 3.5 cm petri dish lid with hole 1.5 cm in diameter
(picture)
- Silicon or vaselin (at high temperature vaselin melts)
Procedure:
1. Cut coverslip to have a 10x6 mm.
2. Cut two peaces of sckotch 5x5 mm.
3. Attach the sckotch to the entire coverslip.
4. Attach the small peace of coverslip above the sckotch as shown in the
picture.
5. Put silicon or vaselin ring around the hole at outer side of the Petri dish lid
(picture).
6. Attach your coverslip to silicon as shown in
picture.
III. Making needles.
Equipment and Materials:
- Puller (we use model PN-30, Narishige, Japan)
- Borosiicate glass capillaries with filament, outer diameter 1.2 or 1.0 mm (cat: BF120-69-15)
- Box to keep the needles
- Forceps (might be useful)
Procedure:
1. Turn on the puller.
2. Set paramiters (in our case Heater=80; MagnetSub=25; MagnetMain=50).
3. Insert the capillar and push red button.
4. Take off the needles and put in the box.
This is a picture of our puller with pulled needles.
IV. Microinjections.
Equipment and Materials:
- Upright fluorescence microscope equipped with 10x objective
- Micromanipulator.
- Any kind of microinjector (from automatic injector,
e.g. Transjector 5246, Eppendorf, to a simple glass syringe)
- Holder for needles
- Filtered solution for injection in a microtube.
Procedure:
1. Put open side of a needle in microtube with injection solution and feel it
controlling the menisk (picture).
2. Fix the needle in the holder and fix the holder in the micromanipulator.
3. Feel the injection chamber with the suspension of 20-30 oocytes.
4. Set the chamber on the microscope stage
(picture).
5. Brake the tip of the needle and perform microinjections.
6. After the microinjection gently transfer oocytes in a Petri dish with 2-4 ml of sea water.
V. Getting ready for experiment.
Equipment and Materials:
- Binocular microscope equipped with 0.6x-10x objectives
- Pipettes 20, 200, 1000 ul.
- Needle for manipulations made of a fine steel needle and 1.0 ml syringe
(picture).
Procedure:
1. Put in the acquisition chamber 0.5-3 ml of sea water.
2. Gently transfer your oocytes to the chamber.
3. Using the needle for manipulations gently fit oocytes beetwin the two coverslips.
4. Gently transfer the acquisition chamber with oocytes to the microscope stage.
Ho-la! You are ready to start!
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