Oct 19, 2023

Kordower Lab Solution Recipes

DOI
https://dx.doi.org/10.17504/protocols.io.261gedxjjv47/v1
  • 1Arizona State University
  • Team Kordower
Scott Muller
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DOI: https://dx.doi.org/10.17504/protocols.io.261gedxjjv47/v1
Protocol Citation: Yaping Chu, Scott Muller, Jeffrey Kordower 2023. Kordower Lab Solution Recipes. protocols.io https://dx.doi.org/10.17504/protocols.io.261gedxjjv47/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 19, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 89630
Keywords: solutions, ASAPCRN, kordower lab solution recipes kordower lab solution recipe, lab
Abstract
Kordower Lab Solution Recipes
Attachments
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Kordower Lab Solutio...
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Materials
Gelatin Type A Chromium Potassium Sulfate
Sucrose Ethylene Glycol Sodium phosphate monobasic (NaH2PO4) Sodium phosphate dibasic (Na2HPO4)
NaCl Trizma base Trizma hydrochloride Triton X-100 Paraformaldehyde 10 Molarity (M) NaOH Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O) Sodium phosphate dibasic heptahydrate (Na2HPO4 · 7H2O) Picric Acid Solution
Slide Subbing Solution
Warm 500 mL of 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 g Chromium Potassium Sulfate
Cryoprotectant Solution
In a 4000 mL beaker, begin with 2000 mL of 0.2 Molarity (M) Phosphate-Buffered Saline (PBS), 7.2
(or 2000 mL dH2O + 2.8 g NaH2PO4 + 10.9 g Na2HPO4 + 18.5 g NaCl, 7.2 )
Add and mix until disolved:
  • 1200 g Sucrose
  • 1200 mL Ethylene Glycol
Bring to 4000 mL final volume with additional PBS
Phosphate-Buffered Saline (PBS)
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 mL of 1X solution. Adjust 7.2 , if needed.
Tris-Buffered Saline (TBS)
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 mL of 1X solution. Adjust 7.4 , if needed.
Dilution Media (DM)
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 mL of 1X solution. Adjust 7.4 , if needed.
4% Paraformaldehyde Solution (PFA)
17m
Solution A (make in 4000 mL beaker in fume hood)
  1. 160 g paraformaldehyde (weigh in fume hood)
  2. Heat 2000 mL dH2O in microwave for 00:07:00 to 00:10:00 until approximately 65 °C
  3. Add heated dH2O to paraformaldehyde in beaker until filled to 2000 mL
  4. Stir on heated stir plate, maintaining 60 °C to 65 °C (do not exceed 70 °C !!)
  5. Add 10 Molarity (M) NaOH until solution clears (typically 10 mL to 20 mL )
  6. Allow solution to cool to <30 °C , can place in ice water bath to speed cooling
17m
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)
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)

Zamboni's Fixative
17m
Solution A (make in 4000 mL beaker in fume hood)
  1. 160 g paraformaldehyde (weigh in fume hood)
  2. Heat 2000 mL dH2O in microwave for 00:07:00 to 00:10:00 until approximately 65 °C
  3. Add heated dH2O to paraformaldehyde in beaker until filled to 2000 mL
  4. Stir on heated stir plate, maintaining 60 °C to 65 °C (do not exceed 70 °C !!)
  5. Add 10 Molarity (M) NaOH until solution clears (typically 10 mL to 20 mL )
  6. Allow solution to cool to <30 °C , can place in ice water bath to speed cooling
17m
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
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 Room temperature for up to 1 year