Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging

Materials and Reagents

1. MatTek Dishes (35 mm, #1.5 coverslip, 20 mm opening: MATTEK, catalog number: P35G-1.5-20-C )
   
2. ColorpHast pH-indicator-strips with pH range 6.5-10 (Merck, catalog number: 109543 )
   
3. 10-100 and 1,000 μl Gilson MICROMAN^® positive displacement pipette tips (Gilson, catalog numbers: FD10004 , FD10006 )
   
4. 10 cm tissue culture dish (Thermo Fisher Scientific, catalog number: 150350 )
   
5. Cell lifters (Corning, catalog number: 3008 )
   
6. Aluminum foil
   
7. Plastic wrap
   
8. Scintillation vial (Sigma-Aldrich, catalog number: Z190527 )
   
9. 1.5 ml microfuge tubes (Thermo Fisher Scientific, catalog number: AM12400 )
   
10. Dubecco’s Minimal Essential Medium (DMEM) powder, Phenol red (Sigma-Aldrich, catalog number: D2429 )
    
11. NaOH pellets (Sigma-Aldrich, catalog number: S8045 )
    
12. Phosphate Buffered Saline with Calcium and Magnesium (PBS⁺⁺) chilled to 4 °C and at room temperature (GE Healthcare, Hyclone^TM, catalog number: SH30264.02 )
    
13. Rat tail collagen solution (dissolved in 20 mM acetic acid, commercial brands are fine, but in-house preparations are usually cleaner and polymerize faster) at a concentration greater than 5 mg/ml (6 mg/ml used here)
14. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
    
15. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034 )
    
16. Boric acid (powder 99.5%, Sigma-Aldrich, catalog number: B6768 )
    
17. Acetic acid (Sigma-Aldrich, catalog number: 27221-1L ) (chilled to 4 °C, less than 50 ml of 16.65 M is required)
    
18. Atto-488 NHS-ester 1 mg (Sigma-Aldrich, catalog number: 41698 )
    
19. DMSO (Sigma-Aldrich, catalog number: D2650 )
    
20. Sircol Collagen Assay kit (available from Accurate Chemical & Scientific, catalog number: CLRS1000 )
    
21. Slide-A-Lyzer Dialysis cassette (G2) with 20,000 MW cutoff (Thermo Fisher Scientific, catalog number: 87735 )
    
22. 1 M Tris (pH 7.4, KD Medical, catalog number: RGF-3340 )
    
23. NaCl (Sigma-Aldrich, catalog number: S7653 )
    
24. HCl (37%, Sigma-Aldrich, catalog number: H1758-500ML )
    
25. 10x DMEM (see Recipes)
    
26. 10x reconstitution buffer (10x RB; see Recipes)
    
27. 1 N NaOH (500 μl in a microfuge tube; see Recipes)
    
28. 1 N HCl (500 μl in a microfuge tube; see Recipes)
    
29. 50 mM Borate buffer pH 9.0 (see Recipes)
    
30. 5 mg/ml Atto-488 NHS-ester dye in DMSO (see Recipes)
    
31. Sodium Chloride (NaCl), 8% solution (see Recipes)
    
32. 50 mM Tris buffer (see Recipes)
    

Equipment

1. 10-100 and 1,000 μl Gilson MICROMAN^® positive displacement pipette (Gilson, catalog numbers: F148314 , F148180 )
   
2. Lab timer
   
3. 2-4 L beaker
   
4. Ultra-clear 8 x 20 mm centrifuge tubes (Optional: Beckman Coulter, catalog number: 345843 )
   
5. Rectangular ice bucket packed with ice
   
6. Water bath
   
7. Large Magnetic stir bar to large beaker (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z284491 )
   
8. Small Magnetic stir bar for scintillation vial (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z126942 )
9. Stir plate
   
10. Mini centrifuge (i.e., VWR, model: Galaxy Mini )
    
11. Biological hood (any brand)
    
12. Brightfield microscope with 10 and 20x phase contrast objectives
    
13. Cooled centrifuge capable of 20,000 x g and 4 °C (i.e., TOMY, model: MX-307 )
    
14. Lab Rocker (i.e., Denville Scientific, model: 110, catalog number: S2110 )
    
15. Airfuge ultra centrifuge (optional: Beckman Coulter, catalog number: 340400 )
    
16. Laser scanning or spinning disk confocal microscope (i.e., Nikon Instruments, model: A1R or Yokogawa, model: CSU-X1 )
    

Procedure