May 21, 2025
Version 2
Isolation of nuclei from fresh frozen brain sections V.2
- Simona Sarafinovska1,
- Allen Yen1,
- Sneha Chaturvedi1,
- Emma Jones1,
- Kelli McFarland White1
- 1Washington University School of Medicine
Protocol Citation: Simona Sarafinovska, Allen Yen, Sneha Chaturvedi, Emma Jones, Kelli McFarland White 2025. Isolation of nuclei from fresh frozen brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9qjdvzp/v2Version created by Rebecca Chase
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: May 21, 2025
Last Modified: May 21, 2025
Protocol Integer ID: 218705
Keywords: nuclei isolation, iodixanol, fresh frozen brain sections, homogenization, snRNAseq, fresh frozen brain sections this protocol, isolation of nuclei, fresh frozen brain section, isolation with an iodixanol, isolation, nuclei, brain section, iodixanol
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
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
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 | 28.8mL | RT | |
| BSA | 10% | 1% | 3.2mL | -4ºC | |
| RNase Inhibitor | 40U/mL | 0.2U/uL | 160uL | -20ºC |
RNase InhibitorPromegaCatalog #N2515
50% Iodixanol - can be prepped during first spin
maintain at 4 °C
| Reagent | Amount-4 Samples (mL) | Stock Temp | |
| OptiPrep (60% Iodixanol) | 6 | -4ºC | |
| DPBS (Ca2+/Mg2+ Free) | 1 | RT |
25% Iodixanol
maintain at 4 °C
| Reagent | Amount-4 Samples (mL) | Stock Temp | |
| 50% Iodixanol | 2 | -4ºC | |
| DPBS (Ca2+/Mg2+ free) | 2 | RT |
Preparation
Before starting - do the following
Turn on benchtop centrifuge with 2 mL swinging-bucket inserts for spins to 4 °C
Clean 2 mL dounce homogenizer and pestles with bleach, rinse with reagent grade water, wash with RNAseZap, and rinse with Millipore water. Wrap top of dounce homogenizer and bottom of pestles with aluminum foil.
Prepare working solutions
Add pre-chilled 500 µL homogenization buffer to the dounce homogenizer and keep on wet ice.
Slide Scraping
Use cell scraper to remove tissue from the 4 100 µm slides. Minimize OCT contamination.
Note
Place finger behind slide to warm up tissue and make it easier to scrape.
Use glass Pasteur pipette to remove tissue from cell scraper and place into dounce.
Spin to remove tissue and leave pipette in dounce to soak.
After scraping all 4 slides, rinse inside of glass pipettes with 500 µL homogenization buffer to remove any remaining nuclei.
Isolation
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
Carefully aspirate the supernatant. Resuspend each pellet to a final volume of 1 mL in homogenization buffer.
Add 1 mL of 50% Iodixanol to samples.
Expected result
2 mL 25% Sample/Iodixanol layer.
Note
Vortex the iodixanol before pipetting.
Weigh tubes to ensure level.
Centrifuge 10000 x g, 4°C , 30 minutes with minimal acceleration and deceleration ramp rate.
With a wide-bore P1000 tip, remove myelin debris from top layer of tube.
Aspirate supernatant leaving behind nuclei pellet and approximately 500 µL of buffer.
Resuspend with 1.5 mL of Nuclei Wash Buffer. 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
Carefully remove and discard the supernatant.
Resuspend nuclei in 1 mL 1x dPBS.
Pass through 100 µm pluristrainers into new 2 mL lo-bind tube.
Wash filter with 200 µL dPBS to remove any additional stuck nuclei.
Keep on ice.
Remove 10 µL into PCR strip, stain with 10 µL AO/PI (1:1) and pipette mix. Load 20 µL on K2 slide and count with MGI Nuclei with Debris settings.
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.