Materials and Reagents

1. Protective gear (gloves, goggles, masks, lab coat)

2. Deionized water

3. Sodium pentobarbital (Sigma, catalog number: P-010 )

4. 1.5% sodium pentobarbital (in saline)

5. NaCl (Sigma-Aldrich, catalog number: S7653 )

6. Saline (0.9% sodium chloride)

7. NaH₂PO₄·2H₂O (Sigma-Aldrich, catalog number: 71645 )

8. Na₂HPO₄·12H₂O (Sigma-Aldrich, catalog number: 71663 )

9. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )

10. NaOH (Sigma-Aldrich, catalog number: S8045 )

11. Hydrochloric acid (HCl) (Sigma, catalog number: H1758 )

12. Heparin (Sigma, catalog number: H3149 )

13. Sodium azide (Sigma, catalog number: S2002 )

14. 4% paraformaldehyde (PFA, in 0.1 M Phosphate Buffer, pH 7.0-7.4) (see Recipes)

Equipment

1. Chemical fume hood

2. Dissecting forceps (RWD, catalog number: F12018-13 ) (Figure 1)

3. Ophthalmic forceps (RWD, catalog number: F12006-10 ) (Figure 1)

4. Hemostatic forceps (RWD, catalog number: F21002-12 ) (Figure 1)

5. Tissue scissors (RWD, catalog number: S13052-12 ) (Figure 1)

6. Fine scissors (RWD, catalog number: S12005-10 ) (Figure 1)

7. Vascular clamp (RWD, catalog number: R33001-48 ) (Figure 1)

8. Micro spatula (Thermo Fisher, catalog number: 21-101-10 ) (Figure 1)

9. 1 ml and 20 ml syringe (Figure 1)

10. 25- or 27-gauge infusion needle (Figure 1)

11. (Optional) Peristaltic pump (LONGERPUMP, catalog number: BT100-2J )

12. 2.5 L and 5 L beakers

13. Thermometer (measuring range:100 °C, division value:1 °C)

14. Measuring cylinder

15. Analytical balance (SARTORIUS, catalog number: BSA124S )

16. Benchtop vacuum

17. 0.22 μm PVDF filter set (Nalgene, catalog number: 291-4520 )

18. 50 ml conical tube (Thermo Fisher, catalog number: 339652 )

19. Fume hood

20. Microwave

21. Magnetic stirrer with heating device (SCILOGEX, catalog number: MS7-H550-Pro )

Figure 1. Surgical instruments

Procedure