A Simple Staining Method Using Pyronin Y for Laser Scanning Confocal Microscopy to Evaluate Gelatin Cryogels

Materials and reagents

Biological materials

1. StemPro^TM BM mesenchymal stem cell (Gibco, Thermo Fisher Scientific, catalog number: A15652)

   Note: Other mesenchymal stem cells (MSCs) may be used, such as those isolated from the umbilical cord [12] or from other sources.

Reagents

1. Pyronin Y (Alfa Aesar, Brand, catalog number: J61068, lot F18Z037), stable at room temperature

2. Glutaraldehyde 25% solution (Sigma-Aldrich, catalog number: G6257); once opened, you may store it in a refrigerator, tightly sealing the container

3. Sodium borohydride (Sigma-Aldrich, catalog number: 452882)

4. Sodium carbonate (Fisher Scientific, catalog number: S64-500) (any equivalent or higher quality of the same chemicals should work fine)

5. Hydrochloric acid (for pH adjustment) (Fisher Scientific, catalog number: A1445-500) (any equivalent or higher quality of the same chemicals should work fine)

6. 16% paraformaldehyde (Electron Microscopic Sciences, catalog number: 15700); once opened, you may store it in a refrigerator, tightly sealing the container

7. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

8. Fish skin gelatin (Sigma-Aldrich, catalog number: G7041)

9. Alexa Fluor 647 phalloidin (Invitrogen, Thermo Fisher Scientific, catalog number: A22287)

10. Dulbecco’s modified Eagle medium (Gibco, Thermo Fisher Scientific, catalog number: 11995), store at 4 °C

11. Fetal bovine serum (premium) (Gibco, Thermo Fisher Scientific, catalog number: A5670701)

12. Dulbecco’s phosphate-buffered saline (PBS) without calcium and magnesium (Gibco, Thermo Fisher Scientific, catalog number: 14190144)

1. Gelatin cryogel solution (see Recipes)

2. 0.1 M sodium bicarbonate buffer (see Recipes)

3. Pyronin Y solution (see Recipes)

4. 4% Paraformaldehyde solution (see Recipes)

5. 0.5% Triton X-100 solution (see Recipes)

1. Gelatin cryogel solution

   Reagent	Final concentration	Amount (per 1.5 mm thick gel)
   Gelatin (8.6%)	4.3%	3,000 μL
   Glutaraldehyde (25%) *	0.3%	72 μL
   H2O	n/a	2,928 μL
   Total	n/a	6,000 μL

   *Add after mixing other parts.




2. 0.1 M sodium carbonate buffer (pH 9.2)

   Reagent	Final concentration	Amount
   Sodium carbonate	0.1 M	10.6 g (when anhydrous is used)
   HCl	n/a	(add dropwise to adjust the pH)
   Total with H2O	n/a	1,000 mL




3. Pyronin Y solution

   Reagent	Final concentration	Amount
   Pyronin Y	1 mg/mL	1 mg
   ddH2O	n/a	1 mL
   Total	n/a	1 mL

   It is highly recommended to spin briefly (~30 s) at 10,000× g to remove any insoluble pyronin Y particles after dissolving the pyronin Y powder.




4. 4% paraformaldehyde solution

   Reagent	Final concentration	Amount
   16% paraformaldehyde solution	4%	2.5 mL
   PBS	n/a	7.5 mL
   Total	n/a	10 mL

   You can downscale the volume.




5. 0.1% Triton X-100 solution

   Reagent	Final concentration	Amount
   Triton X-100	0.1%	10 μL
   PBS	n/a	9,990 μL
   Total	n/a	10 mL

   You can downscale the volume. It will be fast to dissolve Triton X-100 by using a magnetic stirrer bar in a small beaker. Alternatively, making a 10% Triton X-100 stock solution helps this process. If a 10% stock solution is used, the volume of Triton X-100 should be 100 μL.

Laboratory supplies

1. 1.5 mL or 2.0 mL microcentrifuge tubes

2. Micropipette (P200 and P1000)

3. 1.5 mL tube rack

4. 15 mL tubes

5. 100 mm plastic Petri dishes or square dishes (e.g., Carolina Biological Supply, catalog number: 741470)

6. Gloves

7. Flat-tip forceps

8. 35 mm glass bottom dishes (Mattek, catalog number: P35G-1.5-7-C)

   Note: As long as the thickness is #1.5, any glass-bottom dishes for cellular imaging would be suitable.

9. 1.5 mm spacer glass plates (Bio-Rad, catalog number: 1653312)

10. Short glass plates (Bio-Rad, catalog number: 1653308)

11. Mini-PROTEAN tetra cell casting stand & clamps (Bio-Rad, catalog number: 1658050)

12. Mini-PROTEAN casting stand gasket (Bio-Rad, catalog number: 1653305)

13. (alternative for 10/11) glass tube (the bottom has to be tightly sealed with a removable plug) or plastic disposable syringes (no needle required; the top has to be cut off)

Equipment

1. LSM 880 (Carl Zeiss)

2. 20× objective lens; a high numerical aperture, longer working distance objective lens is strongly recommended, such as Plan-Apochromat 20×/N.A. = 0.8 M27 (Carl Zeiss, catalog number: 420650-9901-000)

3. -20 °C freezer (Thermo Fisher Scientific)

4. UV crosslinker (such as Spectronics Corporation, catalog number: XL-1000) or a cell culture hood equipped with a UV lamp

Software and datasets

1. Fiji/Image J (2.15.1), available without any cost

2. Microsoft Excel (no specific version as long as Office 2003 or newer versions are used)

3. Fluorescence Spectraviewer (Bio-Rad; https://www.bio-rad-antibodies.com/spectraviewer.html), available without any costs. Only required if you want to confirm the compatibility of your microscopic illumination settings.

Procedure