Oct 19, 2023

Public workspaceKordower Lab Solution Recipes

DOI
dx.doi.org/10.17504/protocols.io.261gedxjjv47/v1
  • 1Arizona State University
Scott Muller
Open access
DOI: 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
Abstract
Kordower Lab Solution Recipes
Attachments
Kordower Lab Solutions.docx
Kordower Lab Solutio...
18KB
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 Concentration10 Molarity (M) NaOH Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O) Sodium phosphate dibasic heptahydrate (Na2HPO4 · 7H2O) Picric Acid Solution
Slide Subbing Solution
Slide Subbing Solution
Warm Amount500 mL of dH2O to Temperature50 °C to Temperature60 °C , once at temperature, turn off heat, add and mix until dissolved:
  • Amount1.25 g Gelatin Type A
  • Amount0.125 g Chromium Potassium Sulfate
Cryoprotectant Solution
Cryoprotectant Solution
In a Amount4000 mL beaker, begin with Amount2000 mL of Concentration0.2 Molarity (M) Phosphate-Buffered Saline (PBS), Ph7.2
(or Amount2000 mL dH2O + Amount2.8 g NaH2PO4 + Amount10.9 g Na2HPO4 + Amount18.5 g NaCl, Ph7.2 )
Add and mix until disolved:
  • Amount1200 g Sucrose
  • Amount1200 mL Ethylene Glycol
Bring to Amount4000 mL final volume with additional PBS
Phosphate-Buffered Saline (PBS)
Phosphate-Buffered Saline (PBS)
Make Amount1000 mL 7X concentrate and mix until dissolved:
  • Amount1000 mL dH2O
  • Amount9.72 g Sodium phosphate monobasic (NaH2PO4)
  • Amount38.3 g Sodium phosphate dibasic (Na2HPO4)
  • Amount64.8 g NaCl
Add above concentrate to Amount6000 mL dH2O to make Amount7000 mL of 1X solution. Adjust Ph7.2 , if needed.
Tris-Buffered Saline (TBS)
Tris-Buffered Saline (TBS)
Make Amount1000 mL 7X concentrate and mix until dissolved:
  • Amount1000 mL dH2O
  • Amount6.8 g Trizma base
  • Amount46.3 g Trizma hydrochloride
  • Amount61.4 g NaCl
Add above concentrate to Amount6000 mL dH2O to make Amount7000 mL of 1X solution. Adjust Ph7.4 , if needed.
Dilution Media (DM)
Dilution Media (DM)
Make Amount1000 mL 7X concentrate and mix until dissolved:
  • Amount1000 mL dH2O
  • Amount6.8 g Trizma base
  • Amount46.3 g Trizma hydrochloride
  • Amount61.4 g NaCl
  • Amount3.5 mL Triton X-100
Add above concentrate to Amount6000 mL dH2O to make Amount7000 mL of 1X solution. Adjust Ph7.4 , if needed.
4% Paraformaldehyde Solution (PFA)
4% Paraformaldehyde Solution (PFA)
17m
Solution A (make in Amount4000 mL beaker in fume hood)
  1. Amount160 g paraformaldehyde (weigh in fume hood)
  2. Heat Amount2000 mL dH2O in microwave for Duration00:07:00 to Duration00:10:00 until approximately Temperature65 °C
  3. Add heated dH2O to paraformaldehyde in beaker until filled to Amount2000 mL
  4. Stir on heated stir plate, maintaining Temperature60 °C to Temperature65 °C (do not exceed Temperature70 °C !!)
  5. Add Concentration10 Molarity (M) NaOH until solution clears (typically Amount10 mL to Amount20 mL )
  6. Allow solution to cool to <Temperature30 °C , can place in ice water bath to speed cooling
17m
Solution B (make in Amount4000 mL beaker)
  • Amount2000 mL dH2O
  • Amount13.6 g Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O)
  • Amount80.9 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 Ph7.4 , if needed
Store at Temperature4 °C for 1-2 weeks (ok to use if pH remains the same)

Zamboni's Fixative
Zamboni's Fixative
17m
Solution A (make in Amount4000 mL beaker in fume hood)
  1. Amount160 g paraformaldehyde (weigh in fume hood)
  2. Heat Amount2000 mL dH2O in microwave for Duration00:07:00 to Duration00:10:00 until approximately Temperature65 °C
  3. Add heated dH2O to paraformaldehyde in beaker until filled to Amount2000 mL
  4. Stir on heated stir plate, maintaining Temperature60 °C to Temperature65 °C (do not exceed Temperature70 °C !!)
  5. Add Concentration10 Molarity (M) NaOH until solution clears (typically Amount10 mL to Amount20 mL )
  6. Allow solution to cool to <Temperature30 °C , can place in ice water bath to speed cooling
17m
Solution B (make in Amount4000 mL beaker)
  • Amount1700 mL dH2O
  • Amount6.6 g Sodium phosphate monobasic monohydrate (NaH2PO4 · H2O)
  • Amount94.4 g Sodium phosphate dibasic heptahydrate (Na2HPO4 · 7H2O)
  • Amount300 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 Ph7.4 , if needed
Store at TemperatureRoom temperature for up to 1 year