Nov 18, 2020

Public workspaceIsolation and Fixation of Nuclei from the Mouse Brain for Dip-C

DOI
dx.doi.org/10.17504/protocols.io.bpsxmnfn
  • 1Stanford University
Longzhi Tan
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DOI: dx.doi.org/10.17504/protocols.io.bpsxmnfn
Protocol Citation: Longzhi Tan 2020. Isolation and Fixation of Nuclei from the Mouse Brain for Dip-C. protocols.io https://dx.doi.org/10.17504/protocols.io.bpsxmnfn
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: November 17, 2020
Last Modified: November 18, 2020
Protocol Integer ID: 44599
Protocol materials
ReagentsucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #84097
ReagentKCl (2 M), RNase-freeThermo FisherCatalog #AM9640G
ReagentHEPES (1 M)Thermo FisherCatalog #15630080
Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
ReagentKIMBLE 2mL Glass Dounce Tissue Grinder SetMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8938
ReagentKIMBLE Dounce tissue grinder set 7 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9063
ReagentKIMBLE Dounce tissue grinder set 15 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9938
ReagentKIMBLE Dounce tissue grinder set 40 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9188
Reagent KIMBLE Dounce tissue grinder set 100 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0189
Reagent1M DL-Dithiothreitol solution (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #646563
ReagentBovine Serum Albumin (BSA) Fraction V—Molecular Biology GradeGemini Bio-ProductsCatalog #700-106P
ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
ReagentFalcon 40 µm Cell StrainerCorningCatalog #352340
ReagentCorning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235
ReagentParaformaldehyde 32% (methanol free)Electron Microscopy SciencesCatalog #15714
ReagentC-Chip disposable hemacytometerINCYTOCatalog #DHC-N01
ReagentTriton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443
Reagents
Reagents
Prepare 1.5 M sucrose (Amount40 mL ):
  • Amount20.538 g ReagentsucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #84097 (final: Concentration1.5 Molarity (M) , Concentration51.3 Mass / % volume )
  • Amount40 mL water
  • Heat and vortex to mix.
  • Filter to sterilize.
  • Store at Temperature4 °C .
Prepare Nuclei Isolation Medium 1 (Amount45 mL ; note that Tris is replaced with HEPES):
  • Amount7.5 mL 1.5 M sucrose (final: Concentration250 millimolar (mM) , Concentration8.56 Mass / % volume )
  • Amount562.5 µL ReagentKCl (2 M), RNase-freeThermo FisherCatalog #AM9640G (final: Concentration25 millimolar (mM) )
  • Amount450 µL ReagentHEPES (1 M)Thermo FisherCatalog #15630080 (final: Concentration10 millimolar (mM) )
  • Amount225 µL Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G (final: Concentration5 millimolar (mM) )
  • Amount36.2625 mL water
  • Vortex to mix.
  • Store at Temperature4 °C .
Prepare 1% BSA in PBS (Amount1.2 mL per sample; recipe below for Amount10 mL ):
  • Amount0.1 g ReagentBovine Serum Albumin (BSA) Fraction V—Molecular Biology GradeGemini Bio-ProductsCatalog #700-106P
  • Amount10 mL ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
  • Vortex to mix.
  • Rotate until fully dissolved.
  • Chill TemperatureOn ice .
  • Store at Temperature-20 °C if needed.
Nuclei Isolation
Nuclei Isolation
Chill a ReagentKIMBLE 2mL Glass Dounce Tissue Grinder SetMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8938 (or larger sizes: ReagentKIMBLE Dounce tissue grinder set 7 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9063 , ReagentKIMBLE Dounce tissue grinder set 15 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9938 , ReagentKIMBLE Dounce tissue grinder set 40 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9188 , or Reagent KIMBLE Dounce tissue grinder set 100 mL completeMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0189 ) TemperatureOn ice .
Prepare 1 mM DTT:
  • Amount1 mL water
  • Amount1 µL Reagent1M DL-Dithiothreitol solution (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #646563 (aliquoted and stored at Temperature-20 °C )
  • Vortex to mix.
Prepare Nuclei Isolation Buffer without Triton (Amount6 mL per sample):
  • Amount6 mL Nuclei Isolation Medium 1
  • Amount6 µL 1 mM DTT (final: Concentration5 micromolar (µM) )
  • Vortex to mix.
  • Chill TemperatureOn ice .
Prepare Nuclei Isolation Buffer with Triton (Amount2 mL per sample):
  • Amount2 mL Nuclei Isolation Buffer without Triton
  • Amount20 µL ReagentTriton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443 (final: Concentration0.1 % (v/v) )
  • Vortex to mix.
  • Chill TemperatureOn ice .

Add Amount2 mL ice-cold Nuclei Isolation Buffer with Triton to the homogenizer.
Dounce the tissue with 5 strokes of the loose pestle (A), and 15 strokes of the tight pestle (B).
Transfer the homogenate to a tube.
Centrifuge at Centrifigation100 x g, 4°C, 00:08:00 .
Carefully remove supernatant without disrupting the soft pellet.
Resuspend in Amount2 mL Nuclei Isolation Buffer without Triton.
Centrifuge at Centrifigation100 x g, 4°C, 00:08:00 .
Carefully remove supernatant without disrupting the soft pellet.
Resuspend in Amount2 mL Nuclei Isolation Buffer without Triton.
Filter with ReagentFalcon 40 µm Cell StrainerCorningCatalog #352340 or ReagentCorning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235 .
Fixation
Fixation
10m
10m
To every Amount1 mL of cells, add Amount66.7 µL ReagentParaformaldehyde 32% (methanol free)Electron Microscopy SciencesCatalog #15714 (final: Concentration2 Mass Percent ).
Rotate at TemperatureRoom temperature for Duration00:10:00 .
10m
Add Amount200 µL 1% BSA in PBS.
Invert to mix.
Centrifuge at Centrifigation1000 x g, 4°C, 00:05:00 .
Remove supernatant.
Resuspend in Amount1 mL ice-cold 1% BSA in PBS.
Count with ReagentC-Chip disposable hemacytometerINCYTOCatalog #DHC-N01 , and aliquot to up to 0.5 million cells per tube.
Centrifuge at Centrifigation1000 x g, 4°C, 00:05:00 .
Remove supernatant.
Store at Temperature-80 °C .