Warm 500mLof dH₂O to 50°C to 60°C, once at temperature, turn off heat, add and mix until dissolved:

• 1.25g Gelatin Type A
• 0.125gChromium Potassium Sulfate

In a 4000mLbeaker, begin with 2000mL of 0.2Molarity (M) Phosphate-Buffered Saline (PBS), 7.2

(or 2000mLdH2O + 2.8g NaH2PO4 + 10.9g Na2HPO4 + 18.5g NaCl, 7.2)

Add and mix until disolved:

• 1200g Sucrose
• 1200mL Ethylene Glycol Bring to 4000mL final volume with additional PBS

Make 1000mL 7X concentrate and mix until dissolved:

• 1000mL dH₂O
• 9.72g Sodium phosphate monobasic (NaH2PO4)
• 38.3g Sodium phosphate dibasic (Na2HPO4)
• 64.8g NaCl Add above concentrate to 6000mL dH₂O to make 7000mLof 1X solution. Adjust 7.2, if needed.

Make 1000mL 7X concentrate and mix until dissolved:

• 1000mL dH₂O
• 6.8g Trizma base
• 46.3g Trizma hydrochloride
• 61.4g NaCl Add above concentrate to 6000mL dH₂O to make 7000mLof 1X solution. Adjust 7.4, if needed.

Make 1000mL 7X concentrate and mix until dissolved:

• 1000mL dH₂O
• 6.8g Trizma base
• 46.3g Trizma hydrochloride
• 61.4g NaCl
• 3.5mL Triton X-100 Add above concentrate to 6000mL dH₂O to make 7000mLof 1X solution. Adjust 7.4, if needed.

Solution A (make in 4000mL beaker in fume hood)

1. 160g paraformaldehyde (weigh in fume hood)
2. Heat 2000mL dH₂O in microwave for 0h 7m 0s to 0h 10m 0suntil approximately 65°C
3. Add heated dH₂O to paraformaldehyde in beaker until filled to 2000mL
4. Stir on heated stir plate, maintaining 60°C to 65°C (do not exceed 70°C!!)
5. Add 10Molarity (M) NaOH until solution clears (typically 10mL to 20mL)
6. Allow solution to cool to <30°C, can place in ice water bath to speed cooling

Solution B (make in 4000mL beaker)

• 2000mL dH2O
• 13.6g Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O)
• 80.9g Sodium phosphate dibasic heptahydrate (Na2HPO4 · 7H2O)

Vacuum filter Solution A into Solution B in fume hood using Büchner funnel and flask and 15 cm round filter paper

Adjust to 7.4, if needed

Store at 4°C for 1-2 weeks (ok to use if pH remains the same)

Solution A (make in 4000mL beaker in fume hood)

1. 160g paraformaldehyde (weigh in fume hood)
2. Heat 2000mL dH₂O in microwave for 0h 7m 0s to 0h 10m 0suntil approximately 65°C
3. Add heated dH₂O to paraformaldehyde in beaker until filled to 2000mL
4. Stir on heated stir plate, maintaining 60°C to 65°C (do not exceed 70°C!!)
5. Add 10Molarity (M) NaOH until solution clears (typically 10mL to 20mL)
6. Allow solution to cool to <30°C, can place in ice water bath to speed cooling

Solution B (make in 4000mL beaker)

• 1700mL dH2O
• 6.6g Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O)
• 94.4g Sodium phosphate dibasic heptahydrate (Na2HPO4 · 7H2O)
• 300mL Picric Acid Solution

Vacuum filter Solution A into Solution B in fume hood using Büchner funnel and flask and 15 cm round filter paper

Adjust to 7.4, if needed

Store at 4Room temperature for up to 1 year