TITLE: DiI Labelling with a Paintbrush: a Low-cost Alternative to DiOlistic Labelling in Neurons
AUTHORS: Stephanie J. Huang, Joyce Colussi-Mas, Bart A. Ellenbroek

[DESCRIPTION]
DiOlistic labelling is a high-throughput technique commonly used to label neurons in fixed brain tissue using the fluorescent lipophilic carbocyanine dye, DiI. It labels a large population of neurons in a dense yet distributed pattern, making it ideal for morphological studies. However, DiOlistic labelling typically requires an expensive commercial gene gun. Therefore, our protocol presents a low-cost alternative using materials that are already available in most laboratories (e.g. a plastic 12-well culture plate lid) or easily acquired at a low price (e.g. a paintbrush). We detail the DiI labelling process, including the preparation, delivery, incubation, and post-processing steps. Overall, this protocol labels a large population of neurons in a dense yet distributed pattern and, therefore, is a simple and low-cost alternative to DiOlistic labelling.


[BEFORE_START]
See the guidelines section.

[GUIDELINES]
The step-by-step protocol only describes the DiI labelling process. This guidelines section provides reference information regarding the other procedures.

A typical workflow of the procedures involved: Tissue Preparation (Sectioning & Fixation) → DiI Labelling (DiI Preparation, DiI Delivery, DiI Incubation, & DiI Post-processing) → Tissue Mounting → Image Acquisition → Post-processing (e.g. deconvolution) → Morphological Reconstruction 

Tissue Preparation 
This protocol used fresh, fixed adult rat brain tissue that was prepared by rapidly slicing into 800 µm coronal sections using a custom 3D printed brain slicer and fixed by immersing into 1.5% PFA in PB for 24 h at 4°C. The tissue was then stored in PBS at 4°C until DiI labelling (see the figure below). 
Various tissue preparation methods and sample types are compatible with this method. Depending on the sample type, fixation can be done with intracardial perfusion or by immersion fixation, but generally, lower aldehyde concentrations (1.5% or 2% PFA) are preferred over the traditional 4% PFA as higher concentrations impede DiI diffusion and the quality of labelling (although many protocols still use 4% PFA) (Cheng et al., 2014; Kim et al., 2007; Staffend & Miesel, 2011). 
Most protocols use a vibratome to section the tissue, and most commonly use sections with a thickness of around 200-400 µm. The tissue sections tend to be thicker than traditional IHC methods to ensure the sampling of complete neurons/minimise truncations. Cryostats and freezing microtomes are avoided, as freezing will disrupt the cell membrane. Successful labelling impinges on an intact membrane (Gan et al., 2009). 
Methods for fixed samples: rat (Rasia-Filho et al., 2010), rats & mice (Foster-Olive et al., 2018), non-human primate & mice (Seabold et al., 2010), human (Das et al., 2019), and neuronal cultures (Cheng et al., 2014; Kashiwagi et al., 2019).  
Unfixed samples can also be used (although less commonly). However, they require much shorter DiI incubation times, alternative post-processing procedures, and, of course, a shorter visualisation limit.
Methods for unfixed samples: fish & mice (O'Brien & Unwin, 2006), rat (acute slice preparation) (Hosokawa et al., 1992), & unfixed cultures (Honig & Hume, 1986). 
It is crucial to note that the tissue's surrounding environment, especially during sectioning and fixation, can cause extraneous changes to the dendritic morphology. See the following literature (Bourne et al., 2007; Dehghani et al., 2018; Fiala et al., 2003; Idziak et al., 2023; Kim et al., 2007; Kirov et al., 2004; Korogod et al., 2015; Park et al., 1996; Tao-Cheng et al., 2007).
Try to control for these factors as much as possible. However, if not possible, at least ensure the preparation conditions are systematic and consistent amongst experimental samples and be aware that these artifacts can arise.
 

DiI Labelling 
We strongly recommend reviewing the provided notes and video and conducting a pilot study or at least working with test samples before proceeding to experimental samples.

DiI labelling can be combined with other procedures, such as:
Multi-colour labelling  - Multi-colour labelling can be achieved by combining combinations of DiI, DiO, and DiI as in the original DiOlistic labelling study (see Gan et al., 2000; Gan et al., 2009). 
Immunofluorescence - DiI labelling can be coupled with immunofluorescence techniques; however, some modifications may be needed. Particular care should be taken regarding the permeabilization reagent and tissue thickness (see Matsubayashi et al., 2008; Neely et al., 2009; Staffend & Miesel, 2011). 
Tissue Clearing - Tissue clearing may be helpful for thick optical sections. Many tissue clearing methods are incompatible with lipophilic dyes, like DiI, mainly due to the delipidation steps. However, there are DiI-compatible methods available (see reviews by Richardson & Lichtman, 2015; Tian et al., 2021).
 

Tissue Mounting
For our 800 µm thick brain tissue slices, we developed a temporary mounting method allowing imaging of both sides. We used a glass-bottom imaging dish (ibidi µ-Dish 35 mm, high, #1.5) with a washer-weighted coverslip to minimize the distance between the sample and the coverslip. This setup allowed easy flipping of the tissue for imaging of both sides (see figure below).
We used a mounting media containing a mix of ½ glycerol and ½ PBS with 2.5% DABCO antifade (refractive index (RI) ~1.41, matching the RI of the silicone immersion oil of the objective lens). The mounting media also had a mild clearing effect due to its similarity to fixed brain tissue's RI. 
However, the DiI-labelled tissue was not left in the mounting media for extended periods as glycerol has a dye-bleeding effect on membrane dyes like DiI (Cheng et al., 2014; Murphy & Fox, 2007). Still, many protocols use glycerol-based mounting media (Gan et al., 2009; Kim et al., 2007; Staffend & Miesel, 2011) or commercial glycerol containing mounting medias such as Prolong Gold (Cheng et al., 2014; Seabold et al., 2010) or Vectashield (Trivino-Paredes et al., 2019).
Also note that some mounting media, particularly hard-setting ones, can induce tissue shrinkage and compression, potentially altering the neuronal morphology (see interesting discussion thread). 
Traditional mounting and coverslipping can be used for thinner tissue sections featured in other protocols (sometimes with a spacer in between to avoid crushing the tissue) (Foster-Olive et al., 2018; Heck et al., 2012; Rasia-Filho et al., 2010). 
However, it is crucial to note optimisation of the mounting parameters is paramount to successful imaging, especially when it comes to imaging small structures like dendritic spines (see Banecelin et al., 2021; Dumitriu et al., 2011; Foster Olive et al., 2018; Kashiwagi et al., 2019; Peterson et al., 2015). As such, it is essential to minimise the effect of spherical aberrations as much as possible (Diel et al., 2020; Murray, 2011)! 
 

Image Acquisition, Post-processing, & Morphological Reconstruction 
These topics are beyond the scope of this protocol, however, some relevant resources & literature are listed below.

Neuronal Reconstruction
(Allen Cell Types Database, 2018) Technical White Paper: Cell Morphology And Histology (v.7)
(Allen Institute for Brain Science, 2021) 3D Reconstruction of Neurons in Vaa3D V.3
(Arshadi et al., 2021) SNT: A unifying toolbox for quantication of neuronal anatomy
ImageJ SNT https://imagej.net/plugins/snt
NeuroMorpho.Org https://neuromorpho.org
(Parekh & Ascoli, 2013) Neuronal Morphology Goes Digital: A Research Hub for Cellular and System Neuroscience
(Wu et al., 2004) High-throughput Morphometric Analysis of Individual Neurons

Dendritic Spine Morphology
(Das et al., 2019) DiI-mediated analysis of presynaptic and postsynaptic structures in human postmortem brain tissue
(Dumitriu et al., 2011) High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy
(Foster Olive et al., 2018) Diolistic Labeling and Analysis of Dendritic Spines
Harris Lab Wiki http://tinyurl.com/harrislabwiki 
(Heck et al., 2012) A deconvolution method to improve automated 3D-analysis of dendritic spines: application to a mouse model of Huntington’s disease
(Kashiwagi et al., 2019) Computational geometry analysis of dendritic spines by structured illumination microscopy
(Ofer et al., 2022) Structural Analysis of Human and Mouse Dendritic Spines Reveals a Morphological Continuum and Differences across Ages and Species
(Okabe, 2020) Recent advances in computational methods for measurement of dendritic spines imaged by light microscopy
(Paternoster et al., 2018) The importance of data structure in statistical analysis of dendritic spine morphology
(Pchitskaya & Bezprozvanny, 2020) Dendritic Spines Shape Analysis—Classification or Clusterization? 
(Zaccard et al., 2021) Protocol for live enhanced resolution confocal imaging of dendritic spinule dynamics in primary mouse cortical neuron culture

Both &/or General Neuroscience
Allen Brain Map - Morphological Annotation https://portal.brain-map.org/explore/connectivity/synaptic-physiology/synaptic-physiology-experiment-methods/cell-classification#morphology
(Dickstein et al., 2016) Automatic Dendritic Spine Quantification from Confocal Data with Neurolucida 360 
EBRAINS Tools https://www.ebrains.eu/tools
(Harris & Spacek, 2016) Dendrite Structure 
(MBF Bioscience, 2015) Webinar: Neuronal imaging protocols/techniques for 3D neuron reconstruction https://youtu.be/HczuQjeNcR4?si=GKEHj_Nrnl3jZ7Xi
Mouse Brain Atlas https://labs.gaidi.ca/mouse-brain-atlas
Neuroimaging Informatics Tools and Resources https://www.nitrc.org
Rat Brain Atlas https://labs.gaidi.ca/rat-brain-atlas
SynapseWeb https://synapseweb.clm.utexas.edu

General Imaging & Post-Processing
Artifacts & Aberrations in Deconvolution Analysis https://www.olympus-lifescience.com/en/microscope-resource/primer/digitalimaging/deconvolution/deconartifacts
(Bankhead, 2014) Analyzing fluorescence microscopy images with ImageJ
ImageJ NeuroCyto LUTs https://sites.imagej.net/NeuroCyto-LUTs/luts
(Jacquemet et al., 2020) The cell biologist’s guide to super-resolution microscopy
(Jonkman et al., 2020) Tutorial: guidance for quantitative confocal microscopy
(Jost & Waters, 2019) Designing a rigorous microscopy experiment: Validating methods and avoiding bias. (see the additional resources listed in table 1)
(North, 2006) Seeing is believing? A beginners' guide to practical pitfalls in image acquisition
OpenOcular OE2 Adapter https://www.openocular.com
(Pawley, 2000) The 39 Steps: A Cautionary Tale of Quantitative 3-D Fluorescence Microscopy
(Schermelleh et al., 2010) A guide to super-resolution fluorescence microscopy
(Shaw & Rawlins, 1991) The point-spread function of a confocal microscope: its measurement and use in deconvolution of 3-D data
SVI Microscopy Nyquist rate and PSF calculator https://svi.nl/Nyquist-Calculator
(Wallace et al., 2001) A Workingperson’s Guide to Deconvolution in Light Microscopy

[STEPS]
SECTION: DiI Preparation
1. Make the DiI stock solution (recipe for 40 mL of0.15 mg/mL)
 

SECTION: DiI Preparation
1.1. Combine the following in a falcon tube.
 6 mg of DiI crystals
 40 mL of 100% Ethanol
SECTION: DiI Delivery
7. Remove the fixed tissue slice from the PBS and place it onto a clean, dry surface (e.g., a glass petri dish or a well plate lid). 
SECTION: DiI Incubation
11. Return the tissue slice to the PBS in the 12-well plate. Cover and incubate for 16-24 h at RT in the dark to allow the dye to diffuse along the neuronal arbours. 1200 min Room temperature 

 

SECTION: DiI Post-processing
13. Bring the following solutions to Room temperature 
4% PFA
PBS
DAPI 

 

SECTION: DiI Preparation
3. Prepare the DiI-coated well plate lids (150 µL of stock solution per well lid ∴ ~0.0225 mg of DiI per well).
 

 

SECTION: DiI Preparation
3.1. Place a clean, dry lid of a 12-well plate onto a flat surface (interior facing upwards). 
SECTION: DiI Preparation
3.2. Briefly vortex the DiI solution & pipette 150 µLonto each well lid. Make sure the DiI solution does not go over the ledge/condensation rings of the well lid.
SECTION: DiI Delivery
7.1. Using a pair of metal forceps, take a small piece of a shredded Kimwipe and carefully blot the excess PBS from the tissue surface via capillary action. Hold the glass dish and tilt it towards yourself to aid in drawing the liquid away. Avoid directly blotting over the region of interest and overdrying it.

 

SECTION: DiI Preparation
4. Leave to dry overnight on a flat surface in the dark (e.g., in a fumehood or on a bench in a dry room). Avoid disturbing the plate whilst drying; otherwise, it will become uneven. 
  Room temperature 

 

 

SECTION: DiI Preparation
5. Storage - Clasp the dried DiI-coated well plate lid onto the accompanying well plate, wrap it in foil, and store it in a dry, dark cupboard or drawer atRoom temperature until further use (use within 2 weeks). 

 

SECTION: DiI Delivery
9. Rotate the plate 180º. Flip over the paintbrush and drag the unused side of the bristles onto the unused half of the DiI-coated well lid as described in step 8   . Then repeat steps 8.1 and 8.2 (this will be DiI delivery to edge 2 - see figure 6). 

SECTION: DiI Delivery
8.1. Position the paintbrush parallel to the tissue slice, about 2 cm above it, with the bristle tips aligned with the slice's top edge. Use a pair of metal forceps to gently tap the brush's ferrule, dislodging and sprinkling DiI onto the tissue. Between taps, move the brush forward and backward for even dispersion (tap for ~10 s).

 

SECTION: DiI Delivery
8. Take a paintbrush (see materials section & the note below for more information) and load the DiI onto one side of the bristle tips by dragging them across half a DiI well lid in a short, smooth stroke. Try to apply even pressure across the bristles and avoid the edges/condensation ring of the well lid. 

 

SECTION: DiI Delivery
9.1. For more coverage, rotate the slice 90º and then 180º, repeating steps 8, 8.1, and 8.2 (this will be DiI delivery to edges 3 and 4 - see figure 6). Before doing so, either use a new brush or clean the brush by wiping the bristles on a clean Kimwipe and tapping to remove the excess DiI (this is to prevent clumping).
 

 

SECTION: DiI Incubation
12. OPTIONAL: CHECKING THE DENSITY OF LABELLING (1-2 h after DiI delivery)
Carefully remove the labelled slice and mount it onto a glass-bottom imaging dish with plenty of PBS to avoid drying whilst viewing. Afterwards, return to the well and resume the incubation process. 

If the labelling is too sparse, the slice can be relabelled by repeating the processes above. However, the additional handling may damage the tissue, so we typically do not relabel.
Instead, we recommend first becoming proficient with the delivery technique using test samples. Do this by carefully documenting the DiI delivery process and the resulting labelling pattern, then repeating until the desired pattern is consistently obtained. Each time, make small adjustments to the technique and then see how it affects the pattern of neuronal labelling. 
Once mastered, you will be able to perform the DiI delivery with the unaided eye and achieve consistently high-quality labelling. You will not need to perform labelling checks or relabel the tissue.

 

SECTION: DiI Post-processing
14. Under the fumehood, remove the excess PBS from the well containing the labelled tissue and add 3.5 mL of 4% PFA. Incubate for 30 min. 

 

 

SECTION: DiI Post-processing
15. Add 400 µL DAPI solution to the well. Tilt the plate while incubating to ensure the solution fully covers the tissue (otherwise, use more DAPI solution or use smaller volume well plates, e.g. a 24-well plate).

 

SECTION: DiI Post-processing
14.1. Remove the PFA and rinse twice with PBS (1 min per wash).
SECTION: DiI Post-processing
15.1. Remove the DAPI and rinse twice with PBS (5 min per wash).
SECTION: DiI Post-processing
16. Either proceed to storage or tissue mounting. 
For storage, place into a well filled with PBS, cover and wrap in foil, and store at 4°C.
For tissue mounting, refer to the guidelines section for further information.

 

SECTION: DiI Delivery
6. 
 

See the video demonstration & refer to the timestamps in the description bar. 
 

 

SECTION: DiI Preparation
1.2. Vortex the solution intermittently for several minutes until no clumps remain. 
SECTION: DiI Preparation
2. Storage - Decant the prepared DiI solution into a dark/amber glass bottle, seal it, and wrap it with foil to protect it from light. Then, store the bottle at 4 °C until further use.

 

SECTION: DiI Delivery
10. OPTIONAL: LABELLING THE OPPOSITE SIDE OF THE TISSUE 
Repeat on the other side if desired, but make sure to re-moisten the tissue slice with PBS before picking it up. Also, make sure to flip it over onto a clean section of the glass plate to avoid picking up DiI crumbs dropped from the previous steps. 

 

SECTION: DiI Delivery
8.2. Next, vary the brush angle and distance (e.g., hold it perpendicular to the slice) and tap more firmly to dislodge the remaining DiI (tap for ~5 s).
SECTION: Cleaning &amp; Maintenance
17. To clean the paintbrushes, fill a small container with ethanol and dip the DiI-coated bristles into it. Brush against the walls of the container to break up any remaining DiI and drag across a Kimwipe. Repeat this process once, then allow the brush to air-dry for 24 hours before reuse.
SECTION: Cleaning &amp; Maintenance
18. To clean a used DiI-coated well plate lid, pour ethanol over it and allow the DiI to dissolve. Wipe the lid clean with a Kimwipe, and repeat if necessary. Then rinse with ddH2O and air-dry before reuse.
SECTION: Examples
19. Examples of whole neurons
 

Examples of dendritic segments
 

Figures 9a & 9b illustrate the variation in the neuronal labelling patterns and sizes of the DiI crystals/spots produced by this method. Overall, a large population of neurons are randomly labelled.
This method's labelling pattern allows the neuronal morphology to be clearly observed, as depicted by the diverse types in figure 9c. Moreover, the order and origin of the dendritic branches can be easily traced back to the soma, making it suitable for dendritic analyses (see figure 10).
Of course, not every dye spot on the tissue results in neuronal labelling, perhaps due to DiI clumping, failure to incorporate into the tissue, tissue damage, or landing in the neuropil, etc. And not every labelled neuron will be suitable for sampling due to truncation (see figure 10d), insufficient labelling, perpendicular projections or projections beyond the working distance, being obscured by other features &/or cells, etc. 
The sampling criteria will differ depending on the features and analyses of interest. For example, dendritic spine imaging is rather complex and warrants further considerations that are beyond the scope of this protocol (see the literature listed in the guidelines for more information). 
Nevertheless, this method labels a large population of neurons, with plenty suitable for sampling. The following literature provides a good starting point for guidelines on sampling - Dickstein et al. (2016), Dumitriu et al. (2011), and Wu et al. (2004).