Mar 01, 2025
Isolation of nuclei from fresh frozen brain sections
- Simona Sarafinovska1,
- Allen Yen1,
- Sneha Chaturvedi2
- 1Washington University School of Medicine;
- 2Washinton University School of Medicine
Protocol Citation: Simona Sarafinovska, Allen Yen, Sneha Chaturvedi 2025. Isolation of nuclei from fresh frozen brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9qjdvzp/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: January 31, 2025
Last Modified: March 04, 2025
Protocol Integer ID: 119361
Keywords: nuclei isolation, iodixanol, fresh frozen brain sections, homogenization, snRNAseq
Abstract
This protocol is for the isolation of nuclei from fresh frozen brain sections that have been placed on slides. It's main steps consist of homogenization, washing, and isolation with an iodixanol. Once nuclei have been isolated, they should be processed according to the manufacturer's protocol for sequencing.
Materials
Bleach
RNaseZap
Wet Ice
Dry Ice
Low-retention tips
Low-retention tubes
Ultracentrifuge
Swinging-bucket tabletop centrifuge
Dounce homogenizer
Millipore water
Aluminum foil
2mL lo-bind Eppendorf tube
4mL centrifuge tube
15mL low-retention centrifuge tube
1x DPBS
40um filter
Trypan or AO/PI
Protocol materials
RNase InhibitorPromegaCatalog #N2515
In 2 steps
Protease InhibitorRocheCatalog #4693159001
Step 1.1
Before start
IMPORTANT: primate and human brain tissue are potentially infectious. Collect all contaminated
instruments, discarded tissue, and nuclei in a beaker to be bleached at the end
of the experiment. Decontaminated tubes and tips go into a biohazard waste box.
Clean all working surfaces and
pipettes with bleach and RNaseZap. Regularly clean gloves with RNaseZap.
Keep all the solutions and materials on wet ice. Keep the brain samples on dry ice.
All samples should be handled equally at all steps, as different cell types might have nuclei more or less vulnerable to physical dissociation. All pipetting and resuspension steps should be gentle and consistent to avoid nuclei shearing and clumping.
Use low-retention tips and tubes for every step where nuclei are involved.
Solutions
Solutions
Prepare solutions on the day of experiment.
Homogenization Buffer
maintain at 4 °C
| Reagent | Stock | Final | Amount-4 Samples (uL) | Stock Temp | |
| Tris-HCl (pH 7.4) | 1M | 10mM | 120 | RT | |
| KCl | 2M | 25mM | 150 | RT | |
| MgCl2 | 1M | 5mM | 60 | RT | |
| DTT | 1M | 1mM | 12 | -20ºC | |
| RNAse Inhibitor | 40U/mL | 0.2U/uL | 60 | -20ºC | |
| Protease Inhibitor | Dissolve 1 tablet in 600uL nuclease-free water | - | 2 tablets | -20ºC | |
| Nuclease-free water | - | - | Fill to 12mL | RT |
RNase InhibitorPromegaCatalog #N2515 Protease InhibitorRocheCatalog #4693159001
Nuclei Wash Buffer
maintain at 4 °C
| Reagent | Stock | Final | Amount-4 Samples | Stock Temp | |
| DPBS (Ca2+/Mg2+ free) | 1x | 1x | 38.8mL | RT | |
| BSA | 10% | 1% | 3.2mL | -4ºC | |
| RNase Inhibitor | 40U/mL | 0.2U/uL | 160uL | -20ºC |
RNase InhibitorPromegaCatalog #N2515
50% Iodixanol
maintain at 4 °C
| Reagent | Amount-4 Samples (mL) | Stock Temp | |
| OptiPrep (60% Iodixanol) | 17.5 | -4ºC | |
| DPBS (Ca2+/Mg2+ Free) | 3 | RT |
35% Iodixanol
maintain at 4 °C
| Reagent | Amount-4 Samples (mL) | Stock Temp | |
| 50% Iodixanol | 7 | On ice | |
| DPBS (Ca2+/Mg2+ free) | 3 | RT |
Preparation
Preparation
Before starting - do the following
Turn on ultracentrifuge for gradient
Set to 4 °C and ensure that decelerations are set to no brake (RCF: 9999, 30min)
Get Beckman S0410 Rotor (Swinging Bucket) from cold room
Turn on swinging-bucket tabletop centrifuge for spins to 4 °C and ensure that decelerations are set to no brake
Note
Leave note when in use.
Clean dounce homogenizer and centrifuge tubes with bleach, rinse with reagent grade water, wash with RNAseZap, and rinse with Millipore water. Finally, wrap top of tube and bottom of homogenizers with aluminum foil.
Prepare working solutionsgo to step #1
Add pre-chilled 500 µL homogenization buffer to the dounce homogenizer and keep on wet ice.
Isolation
Isolation
Transfer brain tissue to homogenizer.
Homogenize brain tissue. Repeat for all tissue samples.
15 strokes On ice with pre-chilled 'A' pestle and 15 with 'B' grinder.
Note
Minimize bubbles by not removing the pestle from the liquid while homogenizing.
Transfer brain lysate into 2 mL lo-bind Eppendorf tube.
Add 1 mL homogenization buffer to glass homogenizer and transfer remaining lysate to same Eppendorf tube. Keep tube On ice
Swing bucket centrifuge 500 x g, 4°C , 5 minutes
While centrifuging, add 2 mL of 35% Iodixanol to a centrifuge tube (4 ml,
thin-wall polystyrene tube) for each sample.
Note
Vortex the iodixanol before pipetting.
Carefully aspirate the supernatant and resuspend each pellet to a final volume of 1 mL in homogenization buffer.
Add 1 mL of 50% iodixanol to each sample.
Note
Vortex the iodixanol before pipetting.
This will create 25% iodixanol with the nuclei samples.
Pipette the samples up and down with a low retention pipette tip and carefully layer the sample on top of the 35% iodixanol solution.
Note
It is important that the layers do not mix.
Weigh the tubes and adjust the volumes to match the heaviest within 0.1 mg
Centrifuge 10000 x g, 4°C , 30 minutes with minimal acceleration and deceleration ramp rate.
Carefully remove the centrifuge tubes.
The nuclei should be visible as a cloudy layer in the interface of the 25:35% iodixanol.
Remove the nuclei from the interface with a low-retention P1000 tip.
Transfer up to 2 mL to a 15mL low-retention centrifuge tube.
Fill up to 6 mL with Nuclei Wash Buffer and gently mix by inverting or pipetting.
Centrifuge 500 x g, 4°C , 5 minutes
Carefully remove and discard the supernatant.
Resuspend nuclei in 1 mL of Nuclei Wash Buffer.
Centrifuge 500 x g, 4°C , 5 minutes
Resuspend nuclei in 1 mL 1x dPBS and pass them through a 40 µm filter.
Stain with Trypan or AO/PI (1:1) and count on hemacytometer or using a fluorescent microscope or Countess FL Automated Cell Counter.
Check the morphology, size, and absence of nuclei clumps or tissue debris.
Proceed directly to downstream protocol (i.e. nuclei fixation of molecular technology).
Protocol references
Del-Aguila JL, Li Z, Dube U, Mihindukulasuriya KA, Budde JP, Fernandez MV, Ibanez L, Bradley J, Wang F, Bergmann K, Davenport R, Morris JC, Holtzman DM, Perrin RJ, Benitez BA, Dougherty J, Cruchaga C, Harari O. A single-nuclei RNA sequencing study of Mendelian and sporadic AD in the human brain. Alzheimers Res Ther. 2019 Aug 9;11(1):71. doi: 10.1186/s13195-019-0524-x. PMID: 31399126; PMCID: PMC6689177.