Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 144

3. 75% Ethanol.

4. Surgical equipment: fine dissection scissors; two pairs of watchmaker #5 forceps

(sometimes manually sharpened); blunt, fine-curved forceps; serrefine clamp
(1.5 in. or smaller); surgical silk or catgut suture with curved needle (e.g., size
10), 1-mL syringes with 26-gage hypodermic needle.

2.4.2. Preparation of Needles for Microinjection
1. Injection glass needles: with (Narishige #GD-1, Japan) and without (Narishige

#G-1) internal filament.

2. Diamond glass cutter.

3. Needle puller (Narishige).

4. Microforge with 0.22-mm-thick platinum wire (Narishige, Japan).

5. Micropipet grinder (Narishige).

6. Teflon tube linked to a syringe.

7. 10% hydrofluoric acid (Sigma).

8. 100% ethanol.

2.4.3. Mouse Matings
9.

C57B6 mice.

10. Vasectomized males.

11. CBA X C57B6 F1 females.

2.4.4. Isolation of Blastocysts
1. C57B6 females.

2. 70% ethanol.

3. Surgical equipment ( see Subheading 2.8. ).

4. Stereomicroscope.

5. Flush medium: High glucose DMEM, buffered with 20 m M HEPES pH 7.4.

6. 10-mL syringe.

7. 0.60 × 30 mm syringe needle.

7. Transfer pipet.

8. ES medium ( see Subheading 2.3. ).

2.4.5. Preparation of ES Cells for Microinjection
1. ES cells.

2. Feeder cells.

3. 6-cm tissue-culture dishes.

4. ES medium ( see Subheading 2.3. ).

5. 1X PBS ( see Subheading 2.3. ).

6. Trypsin/EDTA ( see Subheading 2.3. ).

7. 10-mL sterile tubes.

2.4.6. Microinjection of ES Cells
1. Microinjection setup: microscope with Hoffman or Nomarski optics (e.g.,

Olympus, Japan or equivalent). Left and right, water-driven micromanipulators