TITLE: Kordower Lab Solution Recipes
AUTHORS: Jeffrey Kordower, Yaping Chu

[DESCRIPTION]
Kordower Lab Solution Recipes

[STEPS]
SECTION: Slide Subbing Solution
1. Warm 500 mLof dH2O to 50 °C to 60 °C, once at temperature, turn off heat, add and mix until dissolved:
1.25 g Gelatin Type A
0.125 gChromium Potassium Sulfate
SECTION: Cryoprotectant Solution
2. In a 4000 mLbeaker, begin with 2000 mL of 0.2 Molarity (m) Phosphate-Buffered Saline (PBS), pH 7.2
(or 2000 mLdH2O + 2.8 g NaH2PO4 + 10.9 g Na2HPO4 + 18.5 g NaCl, pH 7.2)
Add and mix until disolved:
1200 g Sucrose
1200 mL Ethylene Glycol
Bring to 4000 mL final volume with additional PBS
SECTION: Phosphate-Buffered Saline (PBS)
3. Make 1000 mL 7X concentrate and mix until dissolved:
1000 mL dH2O
9.72 g Sodium phosphate monobasic (NaH2PO4)
38.3 g Sodium phosphate dibasic (Na2HPO4)
64.8 g NaCl
Add above concentrate to 6000 mL dH2O to make 7000 mLof 1X solution. Adjust pH 7.2, if needed.
SECTION: Tris-Buffered Saline (TBS)
4. Make 1000 mL 7X concentrate and mix until dissolved:
1000 mL dH2O
6.8 g Trizma base
46.3 g Trizma hydrochloride
61.4 g NaCl
Add above concentrate to 6000 mL dH2O to make 7000 mLof 1X solution. Adjust pH 7.4, if needed.
SECTION: Dilution Media (DM)
5. Make 1000 mL 7X concentrate and mix until dissolved:
1000 mL dH2O
6.8 g Trizma base
46.3 g Trizma hydrochloride
61.4 g NaCl
3.5 mL Triton X-100
Add above concentrate to 6000 mL dH2O to make 7000 mLof 1X solution. Adjust pH 7.4, if needed.
SECTION: 4% Paraformaldehyde Solution (PFA)
6. Solution A (make in 4000 mL beaker in fume hood)
160 g paraformaldehyde (weigh in fume hood)
Heat 2000 mL dH2O in microwave for 7 min to 10 minuntil approximately 65 °C
Add heated dH2O to paraformaldehyde in beaker until filled to 2000 mL
Stir on heated stir plate, maintaining 60 °C to 65 °C (do not exceed 70 °C!!)
Add 10 Molarity (m) NaOH until solution clears (typically 10 mL to 20 mL)
Allow solution to cool to <30 °C, can place in ice water bath to speed cooling
SECTION: 4% Paraformaldehyde Solution (PFA)
7. Solution B (make in 4000 mL beaker)
2000 mL dH2O
13.6 g Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O)
80.9 g Sodium phosphate dibasic heptahydrate (Na2HPO4 · 7H2O)
SECTION: Zamboni&#x27;s Fixative
9. Solution A (make in 4000 mL beaker in fume hood)
160 g paraformaldehyde (weigh in fume hood)
Heat 2000 mL dH2O in microwave for 7 min to 10 minuntil approximately 65 °C
Add heated dH2O to paraformaldehyde in beaker until filled to 2000 mL
Stir on heated stir plate, maintaining 60 °C to 65 °C (do not exceed 70 °C!!)
Add 10 Molarity (m) NaOH until solution clears (typically 10 mL to 20 mL)
Allow solution to cool to <30 °C, can place in ice water bath to speed cooling
SECTION: 4% Paraformaldehyde Solution (PFA)
8. Vacuum filter Solution A into Solution B in fume hood using Büchner funnel and flask and 15 cm round filter paper
Adjust to pH 7.4, if needed
Store at 4 °C for 1-2 weeks (ok to use if pH remains the same)

SECTION: Zamboni&#x27;s Fixative
10. Solution B (make in 4000 mL beaker)
1700 mL dH2O
6.6 g Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O)
94.4 g Sodium phosphate dibasic heptahydrate (Na2HPO4 · 7H2O)
300 mL Picric Acid Solution
SECTION: Zamboni&#x27;s Fixative
11. Vacuum filter Solution A into Solution B in fume hood using Büchner funnel and flask and 15 cm round filter paper
Adjust to pH 7.4, if needed
Store at 4 Room temperature for up to 1 year