2. 10Â phosphate-buffered saline (PBS), pH 7.4 and fibronectin
solution from bovine plasma.
3. Tweezers, filter tips, pipettes, and an aspiration pump.
2.3 Fixation
1. 30% hydrogen peroxide (H 2 O 2 ).
2. 16% paraformaldehyde (PFA), EM grade.
3. 1 M HEPES.
4. pH meter.
5. Vortex mixer and chemical hood.
6. Microcentrifuge tubes.
7. Pipette tips and Pipetman classic™ Starter Kit.
8. Cell membrane marker (wheat germ agglutinin tetramethylrhodamine conjugate).
9. Ibidi μ-dishes.
10. Fluorescence microscope.
11. Fixation solution 1: 4% PFA solution, 200 mM HEPES, and
1Â PBS (see Note 1).
12. Fixation solution 2: 5 mM DTBP solution. Weigh DTBP
powder to make up a 50 mM DTBP stock and dissolve in 1Â
PBS, pH 8.6. Calculate the necessary volume of the stock
solution to get a final concentration of 5 mM DTBP and add
25 mM HEPES in 1Â PBS, pH 8.6 (see Note 2).
2.4 Laser Capture
Microdissection
Molecular Machines & Industries (MMI) CellCut Laser Microdissection and the Software (MMI Cell Tools Software; Ver. 4.3.3.).
2.5 Protein
Extraction
1. Digital dry bath (37, 60, and 100
C).
2. Sonicator.
3. Refrigerated microcentrifuge.
4. Bovine serum albumin standard.
5. RC DC™ Protein Assay Kit II.
6. Truview cuvettes.
7. A
spectrophotometer,
e.g.,
Smartspec™
Plus
Spectrophotometer.
8. Alternatively, samples can be acquired based on the number of
cellular protrusions isolated by LCM, rather than protein assay.
9. Lysis buffer (RIPA): 10 mM Tris–HCl (pH 8.0), 1 mM EDTA,
0.5 mM EGTA, 2% SDS, 1% Triton X-100, 0.1% sodium
deoxycholate, 140 mM NaCl, 100 mM DTT (see Note 3),
1% protease inhibitors (e.g., Halt™ Protease Inhibitor Cocktail, 100Â), and molecular biology grade water.
28
Ana Gordon and Karine Gousset
solution from bovine plasma.
3. Tweezers, filter tips, pipettes, and an aspiration pump.
2.3 Fixation
1. 30% hydrogen peroxide (H 2 O 2 ).
2. 16% paraformaldehyde (PFA), EM grade.
3. 1 M HEPES.
4. pH meter.
5. Vortex mixer and chemical hood.
6. Microcentrifuge tubes.
7. Pipette tips and Pipetman classic™ Starter Kit.
8. Cell membrane marker (wheat germ agglutinin tetramethylrhodamine conjugate).
9. Ibidi μ-dishes.
10. Fluorescence microscope.
11. Fixation solution 1: 4% PFA solution, 200 mM HEPES, and
1Â PBS (see Note 1).
12. Fixation solution 2: 5 mM DTBP solution. Weigh DTBP
powder to make up a 50 mM DTBP stock and dissolve in 1Â
PBS, pH 8.6. Calculate the necessary volume of the stock
solution to get a final concentration of 5 mM DTBP and add
25 mM HEPES in 1Â PBS, pH 8.6 (see Note 2).
2.4 Laser Capture
Microdissection
Molecular Machines & Industries (MMI) CellCut Laser Microdissection and the Software (MMI Cell Tools Software; Ver. 4.3.3.).
2.5 Protein
Extraction
1. Digital dry bath (37, 60, and 100
C).
2. Sonicator.
3. Refrigerated microcentrifuge.
4. Bovine serum albumin standard.
5. RC DC™ Protein Assay Kit II.
6. Truview cuvettes.
7. A
spectrophotometer,
e.g.,
Smartspec™
Plus
Spectrophotometer.
8. Alternatively, samples can be acquired based on the number of
cellular protrusions isolated by LCM, rather than protein assay.
9. Lysis buffer (RIPA): 10 mM Tris–HCl (pH 8.0), 1 mM EDTA,
0.5 mM EGTA, 2% SDS, 1% Triton X-100, 0.1% sodium
deoxycholate, 140 mM NaCl, 100 mM DTT (see Note 3),
1% protease inhibitors (e.g., Halt™ Protease Inhibitor Cocktail, 100Â), and molecular biology grade water.
28
Ana Gordon and Karine Gousset