Jun 04, 2024

Public workspaceFluorescence-activated nuclei sorting (FANS) for single-cell Whole Genome Sequencing (scWGS)

DOI
dx.doi.org/10.17504/protocols.io.81wgbzybygpk/v1
Fluorescence-activated nuclei sorting (FANS) for single-cell Whole Genome Sequencing (scWGS)
  • Ester Kalef-Ezra1,2,
  • Lucia Friscioni1,
  • Dominic Horner1,2,
  • Caoimhe Morley1,
  • George Morrow3,
  • Yanping Guo3,
  • Christos Proukakis1,2
  • 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
  • 3The Flow Cytometry Translational Technology Platform, UCL Cancer Institute, London, UK
Ester Kalef-Ezra
Open access
DOI: dx.doi.org/10.17504/protocols.io.81wgbzybygpk/v1
Protocol Citation: Ester Kalef-Ezra, Lucia Friscioni, Dominic Horner, Caoimhe Morley, George Morrow, Yanping Guo, Christos Proukakis 2024. Fluorescence-activated nuclei sorting (FANS) for single-cell Whole Genome Sequencing (scWGS). protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzybygpk/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: May 15, 2024
Last Modified: June 04, 2024
Protocol Integer ID: 100133
Keywords: ASAPCRN, Sorting, FANS, Immunostaining, Nuclei, Brain, Single-cell, Single-cell Whole Genome Sequencing, scWGS, Single-cell Whole Genome Amplification, scWGA, Aligning Science Across Parkinson’s, ASAP
Funders Acknowledgement:
This research was funded in part by Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research (MJFF)
Grant ID: 000430
This research was supported in part by the MSA Trust
Grant ID: 000430
Abstract
This protocol describes the isolation steps of nuclei from human post-mortem brain samples, immunofluorescence, and nuclei sorting (FANS) for low coverage (<1x) single-cell Whole Genome Sequencing (scWGS) to detect mega-base somatic Copy Number Variations (CNVs).

We have used this protocol to isolate nuclei from the frontal cortex, cingulate cortex, and substantia nigra tissues. However, it can be adapted for nuclei from different body areas, cell culture materials, and/or samples from other species.
Guidelines
Unless otherwise indicated, all the reagents must be kept at 4ºC, and perform the steps in the protocol at 4ºC. To preserve nuclear integrity, all the solutions are supplemented with complete cOmplete EDTA-free Protease Inhibitor Cocktail.
Materials
1. Commercial Reagents:

  • Table 1. Reagents and kit for nuclei isolation using Single Nucleus Isolation Kit (Option 1).
ABCDE
ItemSupplierCatalogue NumberPreparation prior useStorage
MinuteTM Single Nucleus Isolation Kit for Neuronal Tissues/CellsInvent BiotechnologiesBN-020Read kit guidelinesFridge
UltraPure DNase/RNase-Free Distilled WaterThermo Fisher Scientific10977049Aliquot in 7 ml or 50 ml tubesRT
PBS (Phosphate Buffered Saline) 10X Solution (pH 7.4)Fisher Scientific15815418Make 1x with dH2OFridge
Bovine Serum Albumin (BSA)Sigma-AldrichA7030-Fridge
1X PBS with 5% BSA--500 mg of BSA in ~10ml 1X PBS Fridge 

ReagentMinute™ Single Nucleus Isolation Kit for Neuronal Tissues/Cells Invent Biotechnologies incCatalog #BN-020
ReagentPBS (Phosphate Buffered Saline) 10X Solution (pH 7.4)Fisher ScientificCatalog #15815418 ReagentUltraPure&trade; DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977049
ReagentBovine serum albumin (BSA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7030

  • Table 2. Reagents for manual nuclei isolation (Option 2).
ABCDE
ItemSupplierCatalogue NumberPreparation prior useStorage
UltraPure DNase/RNase-Free Distilled WaterThermo Fisher Scientific10977049Aliquot in 7 ml or 50 ml tubesRT
PBS (Phosphate Buffered Saline) 10X Solution (pH 7.4)Fisher Scientific15815418Make 1x with dH2OFridge
50x complete Protease Inhibitor Cocktail EDTA-free (PIC)Roche via Sigma Aldrich4693159001Use 1 tablet in 1 ml dH2O, Aliquot 0.5 ml eachFreezer (20oC)
Triton-X100Sigma AldrichT9287Prepare 10% aliquotRT
ODGM (Optiprep Density Gradient Medium)Sigma AldrichD1556Aliquot 10 ml eachFridge

ReagentUltraPure&trade; DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977049
ReagentPBS (Phosphate Buffered Saline) 10X Solution (pH 7.4)Fisher ScientificCatalog #15815418
ReagentOptiPrep™ Density Gradient MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1556

  • Table 3. Home-Made Reagents for manual nuclei isolation (Option 2).
ABC
ItemSterilization MethodStorage 
1 M MgCl2AutoclaveRT
1 M Tris/HCl pH 8.8AutoclaveRT
1 M Sucrose: FilteredFiltrationFreezer (-20oC)
1 M KClAutoclaveRT
1 mg/ml DAPI (4′,6-diamidino-2-phenylindole)NoFreezer (-20oC)
70% EtOH in dH2ONoRT
  • Table 4. Antibodies for immunodetection.
ABCDEF
AntibodyTypeSupplierCatalogue NumberStock ConcentrationWorking Dilution
anti-NeuN (mouse)*1ryMilliporeMAB3771 mg/mL1/100
anti-Sox6 (mouse)1ryProtein Tech14010-1-A600 µg/mL1/400
anti-NeuN-AF488 (mouse)ConjugatedMilliporeMAB377X1 mg/mL1/100
Anti-Olig2ConjugatedAbcamab2251000.5 mg/ml1/1000
anti-Nurr1-AF488ConjugatedSanta Cruz Biotechnologiessc-376984 AF488200 µg/mlJan-25
AF568 goat anti-mouse2ryLife TechnologiesA110042 μg/ml1/500
AF568 goat anti-rabbit 2ryLife TechnologiesA110112 μg/ml1/500
AF488 goat anti-mouse2ryLife TechnologiesA110012 μg/ml1/500
AF488 goat anti-rabbit 2ryLife TechnologiesA110082 μg/ml1/500
IgG1-AF488Isotype controlMerck Life Science LtdFCMAB310A4
IgG-AF647Isotype controlAbcamab199093


ReagentAnti-NeuN AntibodyMerck Millipore (EMD Millipore)Catalog #MAB377
ReagentAnti-NeuN Antibody, clone A60, Alexa Fluor®488 conjugatedMerck MilliporeSigma (Sigma-Aldrich)Catalog #MAB377X
ReagentRecombinant Alexa Fluor® 647 Anti-Olig2 antibody [EPR2673] (ab225100)AbcamCatalog #ab225100
ReagentNurr1 Antibody (F-5)Santa Cruz BiotechnologyCatalog #376984
ReagentGoat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568Life TechnologiesCatalog #A-11004
ReagentGoat anti-Rabbit IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 Thermo Fisher ScientificCatalog #A11011
ReagentGoat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Life TechnologiesCatalog # A-11001
ReagentGoat anti-rabbit alexaFluor-488 antibodyThermofisherCatalog #A11008
ReagentMilli-Mark™ Mouse IgG1-k, clone MOPC-21, Alexa Fluor® 488 conjugateMerck MilliporeSigma (Sigma-Aldrich)Catalog #FCMAB310A4
ReagentRecombinant Alexa Fluor® 647 Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab199093)AbcamCatalog #ab199093

Note
According to MAB377X description, this antibody works for most neuronal cell types throughout the adult nervous system. However, some neurons fail to be recognized by NeuN at all ages: INL retinal cells, Cajal-Retzius cells, Purkinje cells, inferior olivary and dentate nucleus neurons, and sympathetic ganglion cells and dopaminergic neurons (Mullen et al., 1992; Wolf et al., 1996., Cannon et al., 2009).

2. Reagents for immunodetection:
  • DAPI (4′,6-diamidino-2-phenylindole (stock 1 mg/ml): prepare aliquots of 5 ul each and store at -20°C
  • Goat Serum (Sigma Aldrich G9023): prepare aliquots of 500 ul each and store at -20°C
  • Blocking Buffer: 10% goat serum + 2% PIC in PBS 1x.
Note
For RNA as downstream application, 5% BSA + 0.2 U/µl RNasin in PBS (1x) is suggested as a Blocking Buffer.

  • Optional: Nuclei Storage Buffer: 5 mM MgCl2, 50 mM Tris-HCl (pH 8.8), 166 mM sucrose and 1 mM dithiothreitol (DTT), 1x complete EDTA-free Protease Inhibitor Cocktail.
  • Sorting Buffer: EDTA (final 5 mM) in PBS (1x)

3. General consumables:
  • Flowmi Cell Strainers for 1000uL Pipette Tips, Mesh Size: 70um, Sterile (Fisher Scientific Ltd 15346248)
  • Low binding filtered tips (sterile)
  • Low-binding 1.5ml and 0.2 ml tubes (sterile)
  • Gloves
  • ReagentDNA AWAY&trade; Surface Decontaminant, Surface decontaminant; 8.5 oz. (250mL)Thermo FisherCatalog #7010PK
  • 70% EtOH in dH2O,
  • Cleaning wipes (e.g., Conti Washcloth Dry Brosch Direct PH5959)

4. Additional general consumables and equipment for manual nuclei isolation (Option 2):

  • 1ml syringe without needle (sterile) – Only for manual nuclei isolation (Option 2)
  • PES Syringe filter, 0.2 µm (sterile)
  • PCR cabinet (Here, we used Air Science, Lydiate, UK)
  • PCR cabinet (Here, we used Air Science, Lydiate, UK)
  • ReagentKIMBLE 2mL Glass Dounce Tissue Grinder SetMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8938

5. Equipment:

  • General lab pipettes
  • Pair of forceps and scissors
  • Tissue culture hood for human sample handling
  • Refrigerated centrifuge for 1.5ml tubes that can achieve at least 13000 x g (here used Sigma Aldrich 1 - 14K Refrigerated Micro Centrifuge)
  • Rotator Disk or Tube Roller place in a fridge or cold-room or similar instrument that can allow antibody incubation @4°C with gentle mixing
  • Optional: Haemocytometer to measure/assess nuclei staining
  • Optional: Microscope to measure/assess nuclei staining. Here we used Nikon Eclipse TE300 inverted microscope coupled to a CCD camera - KERN optics)

6. Consumables for nuclei sorting:

  • Reagent96-Well PCR Plate, Non-Skirted (Cuttable), NaturalStarLabCatalog #E1403-0100
  • Reagent VersiCap Mat, 96-well, domed cap stripsThermo ScientificCatalog #15234574
  • ReagentBD FACS™ Accudrop BeadsBecton Dickinson (BD)Catalog #345249
  • ReagentPBS, pH 7.4 (flow cytometry grade)Thermo FisherCatalog #A1286301
  • ReagentAntibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062

7. Equipment for nuclei sorting:

  • BD FACS Aria Fusion sorter (BD Biosciences)
  • Plate centrifuge

  • Table 5. Optical Filters used for sorting using BD FACS Aria Fusion.
ABC
LaserDiva Parameter NameFluorophore(s)
405nm VioletV 450/50DAPI
488nm BlueB 530/30NeuN-AF488, IgG-AF488, Nurr1-AF488
633nm RedR 670/30Olig2-AF647, IgG-AF-647
560nm Yellow-GreenYG 582/15IgG-AF568
8. Reagents for Single-Cell Whole Genome Sequencing and library preparation for Illumina sequencing:

  • Single-cell Whole Genome Amplification. Here we used Takara PicoPLEX® Single Cell WGA Kit R300672)
  • ReagentLow TE BufferInvitrogen - Thermo FisherCatalog #12090-015
  • DNA Library preparation kit. Here we used Agilent SureSelect Enzymatic Fragmentation Kit (5191-6764) and SureSelect XT HS2 DNA Library Preparation Kit (G9985A) with automation using Agilent Bravo. We have also used Illumina DNA PCR-Free Prep, Tagmentation, 96 Samples (20041795) in combination with IDT for Illumina DNA/RNA UD Indexes, such as Set A, Tagmentation (96 Indexes, 96 Samples (20027213). However, other methods can also be used.
Safety warnings
Attention
Please follow the Safety Data Sheets (SDS) for all reagents for safe handling and safety hazards.
Before start
In both cases before use: Clean Human Tissue handling hood with Concentration0.2 Molarity (M) NaOH, 10% Presept, 70% EtOH, and dH2O.
In both cases prior use: Clean Human Tissue handling hood with Concentration0.2 Molarity (M) NaOH, 10% Presept, 70% EtOH and dH2O.
Step case

Single Nucleus Isolation Kit
41 steps

Transfer all materials needed and handle human brain samples carefully in ahuman handling hood.
Note
Critical Notes:
- The Invent Biotech BN-020 kit was initially created for customers to avoid sorting. According to the manufacturer, the kit works with as little as 1 mg of neuronal tissue or cells. However, in our case, we adapted it to isolate nuclei faster and more consistently than the manual method, which requires hands-on preparation of the buffers to be used. Due to our technical needs for cell-type selection and the difficulty of weighing very small amounts of frozen tissue, we use a minimum of 10 mg (usually 10-30mg) of frozen post-mortem brain tissue. Furthermore, as our downstream aim is to do single-cell whole genome amplification of selected nuclei, the use of this kit is followed by nuclei immunostaining and sorting. Due to that, we use the BN-020 kit according to manufacturer instructions with minor modifications.
- According to the Invent Biotech protocol, the Buffer B steps (here 6.9 - 6.13) are optional, but we use them to prepare the cleanest nuclei population possible.
- All centrifugation steps in this part of the protocol can be performed at TemperatureRoom temperature ..

Clean pestles before, between, and after use withConcentration0.2 Molarity (M) NAOH, 10% Presept and dH2O and let them air-dry before use.

Prepare 5% BSA in PBS (1x) and store at Temperature4 °C .

Prepare PIC (1x complete EDTA-free Protease Inhibitor Cocktail) and store in the freezer or remove an aliquot from the freezer and place it TemperatureOn ice .

Use the Invent Biotech BN-020 kit according to manufacturer instructions with minor modifications as follows:

Cut small brain pieces of tissue of interest in pre-weighed tubes and weigh the tissue on a scale aiming for Amount10 mg - Amount30 mg of each tissue/donor depending on the downstream needs.

Note
- The kit can be used for smaller starting material, but we used aminimum 10 mg in order to be able to scale it properly and have enough nuclei for the downstream application.
- If a larger volume of starting material is needed to avoid potential liquid retention in the filter, we suggest splitting the brain pieces into different tubes for nuclei isolation. Then, pool the nuclei together in the same tube before immunostaining.

Add Amount200 µL of cold buffer A in each tube containing a tissue and place it TemperatureOn ice .

Note
From now on, place tubes TemperatureOn ice , if not stated differently.

Pipetting
Homogenize the tissue using the pestle provided by grinding gently with twisting force 50-60 times. Then, add Amount500 µL cold buffer A to the tube and continue to grind 20-30 times.

Incubate the tube TemperatureOn ice for Duration00:05:00 and carefully transfer homogenate to a filter (column) in the collection tube (avoid larger debris that sink to the bottom of the tube).

5m
Incubation
Incubate the tube with the cap open at Temperature-20 °C for Duration00:07:00 .

Note
According to the manufacturer protocol, the incubation time can vary between Duration00:05:00 -Duration00:10:00 .

7m
Incubation
Cap the filter and immediately centrifuge at Centrifigation13000 x g, 00:00:30 .

30s
Centrifigation
Discard the filter (column) and resuspend the pellet by pipetting up and down gently ,10-20 times.

Note
- Try to avoid lipids that attach to the wall of the tube.
- If there is a liquid retention in the filter reduce the amount of starting material by half.

image.png
Figure 1. Sample after centrifugation with the filter column.

Centrifuge at Centrifigation600 x g, 00:05:00 .

Note
The pellet may not be obvious as these are isolated nuclei and it may be in the side close to the bottom of the tube.

5m
Centrifigation
Pour out the supernatant and resuspend the pellet in Amount200 µL PBS with 5% BSA that will be overlaid on top of buffer B in the next step.

Add Amount1 mL cold buffer B to a 1.5 ml eppendorf tube.

Note
Remove bubbles if present.

Pipetting
Carefully overlay the Amount200 µL nuclear suspension on top of buffer B by slowly expelling the nuclear suspension against the wall of the tube.

Centrifuge the tube at Centrifigation1000 x g, 00:10:00 . After centrifugation, cellular debris, oil, and myelin will stay on the top (white-milky layer). The purified nuclei are found in the pellet.

Note
The nuclei pellet may not be visible. This depends on the brain region used.

10m
Centrifigation
Carefully remove the milky layer by withdrawing it into a 1 ml pipette tip and discard the rest of the supernatant.

Pour out the remaining buffer B, leaving Amount50 µL in the bottom of the ultracentrifuge tube (as it contains the nuclear fraction).

Resuspend the pellet in Amount50 µL - Amount200 µL PBS containing Blocking Buffer.

Note
Ensure to rinse the wall of the tube to collect all nuclei.

Nuclei immunostaining
Nuclei immunostaining
2h

Note
  1. When discarding the supernatants after each centrifugation step, leave the last Amount50 µL at the bottom.
  2. Resuspend in same volumes as in prior step to keep the final volume consistent between the steps, but on the last centrifugation step of this section increase the volume to have at least Amount100 µL in each tube.

Incubate nuclei in blocking buffer for Duration00:30:00 at Temperature4 °C in a rotating wheel or in falcon tubes in a tube roller.

30m
Incubation
Add 1ry antibodies or conjugated antibodies directly to the blocking buffer.
Pipetting
Incubate the samples for at least Duration00:30:00 at Temperature4 °C with gentle shaking.

Note
  1. Alternatively, incubation can be done for Duration01:00:00 or over-night.
  2. Prepare negative control for the immunostaining, e.g., split the sample to Amount150 µL with 1ry antibodies and Amount50 µL without 1ry antibodies as negative control.

30m
Incubation
Pellet nuclei at Centrifigation800 x g, 4°C, 00:10:00 and carefully discard supernatant.

10m
Centrifigation
Resuspended in pre-chilled blocking buffer for a gentle wash.
Re-pellet nuclei at Centrifigation800 x g, 4°C, 00:10:00 and discard supernatant.

10m
Centrifigation
Add 2ry antibodies (e.g., AF488 1:500) and DAPI (0.1 µg/ml) in blocking buffer and incubate for at least Duration00:30:00 at Temperature4 °C with gentle shaking.

Note
  1. If conjugated antibodies were used in step 10, skip this step.
  2. DAPI concentration may need to be optimized based on sample and sorter used.

30m
Pipetting
Wash nuclei again with blocking buffer.
Wash
Pellet nuclei at Centrifigation800 x g, 4°C, 00:10:00 and carefully discard the supernatant. Resuspend nuclei in sorting buffer and proceed to filtering using Flowmi Cell Strainers in clean 1.5 mL eppendorf tubes.

Note
If the sorting is scheduled for another day, resuspend nuclei in Nuclei Storage Buffer and store in the fridge for up to 2 weeks. The day of sorting, re-stain the nuclei with DAPI and filter sterilize.

10m
Centrifigation
Nuclei sorting (FANS)
Nuclei sorting (FANS)
Prepare 96-well collection that are compatible with your lab PCR machine by placing Amount5 µL of TE Buffer in each well and seal very carefully using VersiCap Mat strips.

Note
We use 96-well cuttable plates (Starlabs E1403-0100), but other plates such as low bind 96-well plates are recommended.

Transfer nuclei and collection plates on an ice-bucket to the sorting facility.

Note
In our case, we use BD FACSAria Fusion Cell Sorter instrument at the UCL Cancer Institute Flow Cytometry facility.

Acquire small amounts of nuclei from all samples and perform a batch analysis to assess the sample quality and identify the targeted populations for sorting.

Note
  1. Use negative control samples as threshold references.
  2. If possible, keep a consistent gating position across the samples from different donors.
  3. Select gating parameters to isolate the singlets from the overall detected particles by selecting forward (FSC) and side scatter (SSC), FCS single cell gate and SSC single cell gate. Then select the nuclei by their DAPI expression.
  4. From the nuclei population (DAPI+), apply further gating parameters based on the antibodies used.

image.png


image.png
Figure 3. Examples of the gating profiles when sorting human post-mortem nuclei from cingulate cortex stained with NeuN antibodies (A) isolated manually or (B) by using the (B) BN-020 kit. The FCS files were analysed on FlowJo.

image.png
Figure 4. Examples human post-mortem nuclei (A) Cingulate cortex and (B) substantia nigra tissues stained with the conjugated antibodies (A) NeuN-AF488 or (B) Olig2-AF647 (left panel) or their isotype controls (right panel).

Centrifuge the collection plates for Duration00:00:10 at low speed to ensure reagent at bottom.

10s
Centrifigation
Sort the single-nuclei of interest into the centrifuged 96-well collection plates.

Note
Before single cell sorting, use Accudrop beads for a test sort to evaluate the position of the plate and to ensure the sorted cells will be deposited into each well accurately in the middle.

Cap the collection plates carefully to ensure all sorted cells are merged in the reagent and not attached to the side of tube.
Centrifuge the collection plates and place them TemperatureOn ice for transportation to the laboratory.

Note
You can directly proceed to Section 4 or store the plates at Temperature-70 °C .

Single-nucleus Whole Genome Amplification and library preparation
Single-nucleus Whole Genome Amplification and library preparation
Thaw collection plates TemperatureOn ice .

Centrifuge the collection plates briefly prior use.
Centrifigation
Perform single-nucleus Whole Genome Amplification according to Takara’s guidelines.

Note
We observe approximately 85-100% amplification success rate depending on the sample/donor used.

Prepare compatible libraries for sequencing, such as Agilent SureSelect XT HS2, Illumina DNA PCR-Free prep or other DNA library preparation kit.
Perform library pooling and sequence the libraries.
Protocol references
This protocol was adapted from:

Option A. Single Nucleus Isolation Kit:
Adapted from Invent Bioscience kit BN-020 (Invent Biosciences protocol) manual.

Option B. Manual nuclei isolation and immunostaining:
1. Reed PJ, Wang M, Erwin JA, et al. (2017) Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity. In: Frade J., Gage F. (eds) Genomic Mosaicism in Neurons and Other Cell Types. Neuromethods, vol 131. Humana Press, New York, NY.
2. Wierman MB, Burbulis IE, Chronister WD, Bekiranov S, MJ MC (2017) Single cell CNV detection in neuronal nuclei. In: Springer (ed) Genomic Mosaicism in Neurons and Cell Types (editors: Frade JM, Gage FH). New York, USA: Humana Press, Springer Nature; ISBN 978-1-4939-7279-1.
3. Perez-Rodriguez D, Kalyva M, Santucci C, Proukakis C (2022) Somatic CNV Detection by Single-Cell Whole-Genome Sequencing in Postmortem Human Brain. In: Methods in Molecular Biology: Alzheimer’s disease. Vol. 2561, Jerold Chun (Ed).
4. Ester Kalef-Ezra, Diego Perez-Rodriguez, Christos Proukakis (2023) Manual isolation of nuclei from human brain using CellRaft device and single nucleus Whole Genome Amplification. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzjjov8j/v1.

Other references:

1. Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development. 1992;116(1):201-211. doi:10.1242/dev.116.1.201.
2. Wolf HK, Buslei R, Schmidt-Kastner R, et al. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem. 1996;44(10):1167-1171. doi:10.1177/44.10.8813082.
3. Cannon JR, Greenamyre JT. NeuN is not a reliable marker of dopamine neurons in rat substantia nigra. Neurosci Lett. 2009;464(1):14-17.