Mar 18, 2026
Nuclei Extraction from OCT-Embedded Brain Blocks or Liquid Nitrogen Vapor-Frozen GBM Brain Tissue
- Osama Al Dalahmah1
- 1Columbia University Irving Medical Center
- CUMC_Glial_Pathology_Lab
Protocol Citation: Osama Al Dalahmah 2026. Nuclei Extraction from OCT-Embedded Brain Blocks or Liquid Nitrogen Vapor-Frozen GBM Brain Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld463nl5b/v1
Manuscript citation:
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: March 17, 2026
Last Modified: March 18, 2026
Protocol Integer ID: 313383
Keywords: frozen gbm brain tissue, nuclei extraction from oct, gbm brain tissue, nuclei extraction, isolation of nuclei, rnase inhibitor buffer, iodixanol density purification, nuclei, nucleus application, liquid nitrogen vapor, final resuspension in pb
Abstract
**Purpose. This protocol describes isolation of nuclei from OCT-embedded or liquid nitrogen vapor-frozen GBM brain tissue using Dounce homogenization, serial filtration, low-speed centrifugation, iodixanol density purification, and final resuspension in PBS/BSA/RNase inhibitor buffer for downstream single-nucleus applications.
Guidelines
- Both OCT-embedded tissue and liquid nitrogen vapor-frozen tissue are compatible with this protocol.
- Flowmi filter tips may reduce yield relative to BD strainers.
- The estimated duration is approximately 2.5 hours for two samples without a density gradient and approximately 4 hours with a density gradient.
Materials
**Reagents**
- DMSO (Sigma, Cat# 472301)
- BSA (Jackson ImmunoResearch, Cat# 001-000-162)
- Triton X-100 (Thermo Scientific, Cat# 327372500)
- DAPI (Thermo Scientific, Cat# 62248)
- Propidium iodide (Invitrogen, Cat# P3566)
- Hoechst 33342 (Invitrogen, Cat# H3570)
- Halt Protease Inhibitor Single-Use Cocktail 100× (Thermo Scientific, Cat# 1860932)
- Ambion RNase Inhibitor (Invitrogen, Cat# AM2684)
- SUPERase-In RNase Inhibitor (Invitrogen, Cat# AM2696)
- DTT
- OptiPrep / 60% iodixanol (Sigma Cat# D1556)
- Nuclease-free water
- Nuclease-free PBS
- Trypan Blue 0.4% (Gibco, Cat# 15250061)
**Equipment**
- Hemocytometer or Countess II
- Fluorescence/light microscope
- Liquid nitrogen flask
- Dounce tissue grinders (DWK Life Sciences Wheaton™ Dounce Tissue Grinders cat#06-434)
- 1.5 mL microcentrifuge tubes (Eppendorf)
- 2.0 mL Eppendorf Safe-Lock microcentrifuge tube, natural, round bottom (Eppendorf Cat# EP022600044-100EA – sourced from Sigma). These are used for density gradient steps
- BD Falcon filter tubes with strainer caps (Corning, cat# 352235)
- Flowmi strainers (Bel-Art, cat# H13680-0400 (40µM) and cat# H13680-0700 (70µM))
- Refrigerated centrifuge set to 4 C
- 10 cm Petri dish
- Dissection tools (optional)
Troubleshooting
Before start
Perform all steps on ice unless noted otherwise. Prepare RNase inhibitor-containing buffers fresh on the day of use.
Buffer and Solution Recipes
Nuclei isolation medium #1 (NIM1); prepare 15 mL total
- 1.5 M sucrose: 2500 µL
- 2 M KCl: 187.5 µL
- 1 M MgCl2: 75 µL
- 1 M Tris pH 8.0: 150 µL
- Nuclease-free water: 12087.5 µL
Nuclei isolation medium #2 (NIM2); prepare fresh and keep on ice; 5 mL total
- NIM1: 4895 µL
- 1 mM DTT: 5 µL
- 100× protease inhibitor: 50 µL
Homogenization buffer; prepare fresh and keep on ice; 1.5 mL total
- NIM2: 1450.5 µL
- RNase inhibitor, 40 U/µL: 15 µL
- SUPERase-In, 20 U/µL: 15 µL
- 10% Triton X-100: 15 µL
- Propidium iodide: 1.5 µL
- Hoechst: 1.5 µL
Iodixanol medium (IDM); prepare 15 mL total
- 1.5 M sucrose: 2500 µL
- 1 M KCl: 2250 µL
- 1 M MgCl2: 450 µL
- 1 M Tris pH 8.0: 900 µL
- Nuclease-free water: 8900 µL
50% iodixanol; prepare 15 mL total
- 60% iodixanol: 12500 µL
- IDM: 2500 µL
29% iodixanol; prepare 15 mL total
- 60% iodixanol: 7250 µL
- IDM: 7750 µL
PBS+ resuspension buffer
- PBS supplemented with 1% BSA
- Add RNase inhibitor to 0.2 U/µL final concentration
Nuclei storage buffer
- 1.5 M sucrose: 0.855 g - final concentration(166.5 mM)
- 1 M MgCl2: 50µl - final concentration(5 mM)
- 1 M Tris buffer, pH 8.0: 500µl -final concentration(10mM)
- Nuclease-free water: 14,450µlbd falcon
Procedure
Prepare all buffers. For the homogenization and nuclei isolation buffers, use 1.5-2X buffer volume per sample. Any RNase inhibitor-containing buffer should be made fresh on the day of use.
Prepare wet ice and dry ice.
Chill all dissection tools and the Petri dish on ice.
Clean tools and work surfaces with RNaseZap and ethanol as appropriate.
Prepare counting tubes containing Trypan Blue, PBS, and DAPI.
For each sample, prepare a density gradient tube with 500 uL 50% iodixanol and a top layer of 29% iodixanol; keep on ice.
Prepare PBS + 1% BSA plus RNase inhibitor, homogenization buffer, and pre-fill Dounce homogenizers with 1 mL homogenization buffer.
Pre-cool a 10 cm Petri dish on dry ice for 15 minutes and ensure the centrifuge is at 4 C.
Keep tissue on dry ice until the stage is ready.
Transfer OCT-embedded or liquid nitrogen vapor-frozen tissue to the cold stage.
Trim excess OCT and dissect approximately two cortex wedges ~ 0.5 x 0.4 x 0.3cm – maximal diameter is less than 0.5 cm for optimal results; cut each wedge into two pieces and transfer to the Dounce homogenizer. Minimize white matter carryover.
Homogenize using 15 loose strokes followed by 15 tight strokes. The number of strokes can be optimized on a case-by-case basis.
Add 0.5 mL homogenization buffer to each homogenizer.
Filter homogenate through BD Falcon strainer tubes in 150 uL aliquots, pipetting slowly at a shallow angle.
Tap tubes gently to assist flow-through.
Optionally repeat filtration once. 70 µM Flowmi strainers may be used as an alternative, although they can reduce recovered volume.
Centrifuge filtered homogenate at 1000 x g for 10 minutes at 4 C.
Remove supernatant and resuspend in 1000 uL homogenization buffer. Remove 10 uL for counting.
Filter again using BD strainers or 40 µm Flowmi tips.
Centrifuge at 1000 x g for 10 minutes at 4 C.
Remove supernatant and gently resuspend pellet in 250 uL homogenization buffer.
Prepare HB+ by adding 25 to 30 uL Trypan Blue 0.4% to 0.5 mL homogenization buffer. Propidium iodide may also be added to improve pellet visibility.
Resuspend the pellet in 260 uL HB+ and take 10 uL for counting.
Add an equal volume of 50% iodixanol to the nuclei suspension to generate a 25% iodixanol nuclei mix.
Slowly layer this sample onto the pre-made gradient tube containing a 50% iodixanol bottom cushion and 29% iodixanol upper layer in a 2ml Eppendorf tube.
Centrifuge at 18000 x g for 23 minutes at 4 C.
Recover nuclei from the interface between the 29% and 50% iodixanol layers. Nuclei may appear as a pellet or sheet; proceed even if a clear pellet is not visible.
Carefully remove the upper blue layer, then the 29% iodixanol, and finally the 50% cushion while protecting the nuclei interface.
Gently resuspend purified nuclei in 50 to 200 uL PBS+ (PBS + 1% BSA + 0.2 U/uL RNase inhibitor).
Filter one half of the nuclei stock through a 40 um Flowmi tip after 1:1 dilution with PBS+.
Remove 10 uL for counting.
Count nuclei using a hemocytometer or Countess II and repeat counts until the standard deviation is below 25% if possible.
Adjust nuclei concentration to 700 to 1200 cells/uL.
Submit more than 20 uL to the single-cell core for 10x Genomics processing.
(Optional): Freeze remaining nuclei in 10% DMSO plus PBS + 1% BSA + RNase inhibitor in liquid nitrogen or use NSB for long-term storage. The utility of frozen nuclei varies depending on the downstream application. In our experience, they are not suitable for a second round of 10x genomics application.