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Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Published: March 31, 2022 doi: 10.3791/63679


Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.


Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.


In recent years, the knowledge of astrocyte function and how they interact with neuronal circuits has increased dramatically. Astrocytes can influence plasticity1,2, assist in neuronal postinjury recovery3,4, and even induce de novo neuronal potentiation, with recent studies exhibiting the importance of astrocytes in memory acquisition and reward, previously regarded as purely neuronal functions5,6,7. A feature of particular interest in astrocyte research is the spatial arrangement of the cells, which maintain unique spatial organizations in the hippocampus and other brain structures8,9,10. Unlike the neuronal dendrites that intertwine between cell somata, hippocampal astrocytes inhabit visually distinguishable territories with slight overlap between their processes, creating distinct domains8,11,12,13. The evidence supporting the participation of astrocytes in neuronal circuits does not support the lack of detailed anatomical description of such populations and the neurons in their domains14.

Viral vector transduction procedures, along with transgenic animals (TG), have been popularized as a toolset to investigate brain structures, functions, and cell interactions15,16. The utilization of different promoters allows the targeting of specific cells according to their genetic properties, activation levels17,18, or projection targets. Different viruses can express different colored fluorophores in different populations. A virus can be combined with the endogenous expression of fluorophores in TG, or TG animals can be used without the need for viruses. These techniques are widely used for neuronal marking, and some labs have started using them with modifications specialized for targeting other cell types, such as astrocytes5,9,19.

The CLARITY technique, first described in 201320,21, enables the study of thick brain slices by making the entire brain transparent while leaving the microscopic structures intact. By combining the two methods-viral vector transduction and tissue clearing-the option of examining the spatial interactions between different cell type populations is now available. Most astrocyte-neuron interaction studies were performed on thin brain slices, resulting in images of incomplete astrocytes due to their large domains, thus radically restricting the number of analyzed cells. The use of the CLARITY technique allows single-cell resolution characterization of cell populations in large-scale volumes simultaneously. Imaging fluorescently tagged cell populations in clear brains does not deliver synaptic resolution but permits thorough characterization of the spatial interactions between astrocytes and a variety of neuronal cell types.

For that reason, we harnessed these state-of-the-art techniques to investigate the properties of astrocytes throughout the dorsal CA1, imaging all lamina (Stratum Radiatum, pyramidal layer, and Stratum Oriens). We measured tens of thousands of astrocytes (with viral penetrance of >96%5), thereby analyzing the information of the entire astrocytic population around CA1. With efficient penetrance of the neuronal markers, we could record the interactions between the entire population of CA1 astrocytes and the four types of neuronal cells-parvalbumin (PV), somatostatin (SST), VIP inhibitory neurons, and excitatory pyramidal cells9.

Several experiments were performed using a combination of fluorescence from TG animals and differently colored viral vectors (all inhibitory cells), while others (excitatory) utilized two viral vectors expressing different fluorophores under different promoters9. This paper presents a detailed protocol, including the tagging of the desired cells in the brain, making the brain transparent using a modified CLARITY procedure, as well as imaging and analyzing complete brain structures, using various procedures and software.

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Experimental protocols were approved by the Hebrew University Animal Care and Use Committee and met the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals.

1. Viral vector transduction

NOTE: Viral vector transduction is used to express fluorophores in the brain.

  1. Use an atlas (e.g., Allen Brain Atlas) to locate the relevant coordination of the target area.
    NOTE: 3D atlases can be found online (e.g., http://connectivity.brain-map.org/3d-viewer).
  2. Using stereotactic surgery, inject the viral vectors into the relevant brain structure. See9 for a detailed protocol.
  3. Wait for 3-6 weeks for fluorophore expression.
    NOTE: A short period of time is enough if only cell bodies need to be marked. A long period of time will be needed if the axonal projections are relevant to the question, as it takes longer for the fluorophores to express in axons, which can be several millimeters long.
  4. Validate the specificity and penetrance of fluorophore expression (both of viral expression and TG animals) beforehand (Figure 1A). Before proceeding with the CLARITY procedure, designate at least one brain for thin slices and make sure the fluorophore expression is both strong and specific to the target cell feature.


NOTE: This method renders the brain transparent within 2-6 weeks.

  1. Perform transcranial perfusion on animals using cold Phosphate-buffered Saline (PBS) followed by 4% Paraformaldehyde (PFA) in PBS. Remove the brain and keep it in PFA overnight at 4 °C in a 50 mL tube or a similar container.
    NOTE: Before transcranial perfusion is performed, the animal must be deeply anesthetized. In the examples presented in this protocol, all animals were anesthetized using Ketamine and Xylazine (90% and 10%, respectively).
  2. Replace PFA with Hydrogel Solution (HS; see Table 1) for 48 h at 4 °C.
    NOTE: Do not allow the materials to become warmer than the refrigeration temperature (4-8 °C) at this stage, or the Hydrogel will polymerize. The HS used in this protocol contains 2% acrylamide, unlike previous protocols suggesting 4%22 or 1%17. The benefit of 2% is detailed in the discussion section. When preparing the HS, work on an ice-cold surface. Store it at -20 °C.
  3. Degassing
    NOTE: The purpose of this stage is to remove all free oxygen from the tissue as O2 interferes with the polymerization process. Any nonO2 gas can be used (e.g., N2, CO2); N2 is recommended.
    1. Transfer the N2 from the tank via a 5 mm (internal) flexible tubing, connected to a 19 G needle at its end (Figure 1B).
    2. Make two small holes (needle-wide) in the cap of the tube: one to introduce the gas and the other to allow the air to leave the tube (Figure 1C).
    3. Attach the pipe from the N2 gas to the tube and replace the gases for approximately 30 min at room temperature (RT).
    4. Remove the pipe and immediately seal the holes with modeling clay on every tube (Figure 1D).
  4. Transfer the degassed sealed tubes to a 37 °C bath for 3.5 h to polymerize the HS, which will become a gel. Be careful not to shake the tubes (Figure 1E).
  5. Extract the brain from the tube and gently remove the polymerized gel from around it using laboratory wipes. Make sure no residual gel remains attached to the surface of the tissue, as it might react with the solution in the following steps, inhibiting the tissue clearing process (Figure 1F).
    NOTE: Because the gel contained PFA, the extraction should be done under a fume hood.
  6. Slice the brain if the question at hand requires only a part of it. Divide it in half or into very thick slices that contain all areas of interest (Figure 1G).
  7. Place the slices in the First Clearing Solution (CS1, see Table 2) in a new container and shake at 70 rpm for 24 h at 37 °C.
  8. Follow the steps described below to transfer the brain from CS1 to the second Clearing Solution (CS2, see Table 2).
    1. Prepare a perforated tube for the brain in advance (Figure 1H).
    2. Preheat CS2 to 40-45 °C in a beaker large enough to contain the tube. Do this on a hotplate with a stirrer. Ensure the temperature does not reach 55 °C to prevent the bleaching of the expressed proteins.
    3. Place the beaker filled with CS2 and the perforated tube on a stirring device (a 2 L beaker in Figure 1I). Set a moderate stirring rate that will cause the liquid to flow without distorting the tissue.
      NOTE: Within 2-6 weeks, the tissue will become transparent (the process starts at the periphery and moves inward toward the central brain structures). The decision as to when the tissue is "clear enough" is up to personal judgment. The brain should be sufficiently transparent for imaging under the microscope without interference (Figure 1J not clear enough; Figure 1K clear enough). Surpassing the point at which the tissue is clear enough may cause loss of tissue rigidity.
  9. Transfer from CS2 into PBST (0.5% Triton X-100 in PBS, see Table 2) in a new container at 37 °C with mild shaking (70 rpm) for 24 h.
    NOTE: In the PBST, the tissue will become whitish (Figure 1L). The clarity will return (and improve) when embedded in the Refractive Index Matching Solution (RIMS, see step 2.14).
  10. Replace the PBST with new PBST. Keep under the same conditions (37 °C with mild shaking) for another 24 h.
  11. Replace PBST again with new PBST and keep at RT for 24 h.
  12. Transfer to PBS at RT for 24 h.
  13. Replace the PBS and leave at RT for an additional 24 h.
  14. Remove the brain from PBS and transfer it to RIMS at 37 °C overnight.
    NOTE: Initially, ripples in the RIMS may surround the brain. This protocol was designed and validated using two specific commercial RIMS (see Table 3). However, the protocol should not differ when using other RIMS (commercial or self-made).
  15. Keep the tissue at RT for another 24 h or until the solution reaches full equilibrium, i.e., when the tissue becomes transparent, and the solution no longer contains any visible ripples (Figure 1M).
  16. If the tissue is transparent, proceed to the chamber preparation. If at this point, the tissue becomes white instead of transparent (due to aggregates of residual SDS molecules), clean the sample again by repeating steps 2.9 and 2.10, and transfer the tissue to CS2 (step 2.8) for a few days, followed by all the steps until step 2.15.

3. Chamber preparation

NOTE: Each sample requires a slide with an imaging chamber in which the sample will be placed.

  1. Place the sample in the middle of the slide.
  2. Using a hot glue gun, create walls at the edges of the slide, almost as high as the tissue. Make sure to leave a small gap (approximately 5 mm) at one of the corners (Figure 2A,B).
  3. Apply 1-2 drops of the RIMS on the sample to keep the upper surface moist and prevent bubbles forming between the coverslip and the tissue.
  4. Immediately after applying the RIMS drops, add the last layer of hot glue to the walls (so that they reach the height of the brain/slice) and progress immediately (while the hot glue is still liquid) to step 3.5.
  5. Seal the top with a coverslip, placing it as evenly as possible on the top of the still-warm hot glue layer (Figure 2C).
  6. Fill the chamber with the RIMS through the gap left in the hot glue walls (Figure 2D,E).
  7. Close the gap with hot glue. Leave no air inside (Figure 2F).
  8. If the hot glue walls extend beyond the borders of the slide, cut the extending edges (Figure 2G).
  9. If the objective used for imaging is immersed, add another 2-3 mm of glue to the walls above the coverslip so that the immersion solution will last for a longer period (Figure 2H).

4. Imaging (confocal or two-photon)

  1. Check whether the stage can hold the chamber, as the thickness of the chamber can reach several millimeters.
    NOTE: For example, the confocal microscope (see the Table of Materials) used in this protocol is equipped with several stages (e.g., circular, rectangular). Whichever stage is chosen to hold the chamber is irrelevant as long as its parameters fit the chamber sizes.
  2. Work with an objective with a sufficient working distance, i.e., a minimal working distance ≥3 mm, as the brain region of interest may be a few millimeters in depth, and the coverslip may not be completely straight as it was placed manually.
    NOTE: As a demonstration, the results presented in Figure 1, Video 1, and Video 2 were obtained under a two-photon microscope using a water immersion 16x objective with a 3 mm working distance and magnification of 2.4 to obtain a 652 x 483 µm field of view and a z-stack with 0.937 µm intervals between the planes.
  3. Perform multiple-area imaging, which may take a few hours or even days. Make sure there is ample free storage in the computer as the size of each image can reach up to tens of gigabits.
    NOTE: CLARITY imaging may result in significantly more image sections in depth. Therefore, the intensities used to capture the expression should be relatively low to prevent overexpression during the image summation.
  4. For immersion objectives, check the liquid routinely while imaging and supplement with additional liquid if needed.
    NOTE: During the imaging of the data presented in Video 3, water was added to the surface of the chamber every 6-8 h to combat evaporation.
  5. After the image has been acquired, view and analyze it using several different software packages.
    NOTE: Recommended software includes Imaris and SyGlass for both visualization and analysis. The precise pipeline for optimized reconstruction of astrocytes using Imaris software has been described previously9. In short, the Imaris features used in this study were Filaments to fully reconstruct cell structures and Spots to extract the position in the space of all the somata.

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Representative Results

Successful clearing of thick brain tissue slices results in a new range of questions that can be asked regarding the properties of large cell populations as opposed to the properties of single cells or neighboring groups of cells. To achieve successful results, one should strictly adhere to the CLARITY protocol, as there is a wide range of parameters that need to be considered to reduce the variance between samples (e.g., percentage of clarity, fluorescence information, swollenness parameters).

Figure 1 describes the entire clearing process from fluorescent protein expression validation through all the stages of the clearing method. Figure 2 describes how to prepare a chamber for the clarified tissue, optimal for imaging under a two-photon or confocal microscope. The materials required in this protocol are extremely inexpensive relative to other protocols of chamber preparation.

Figure 3 presents the cube described in Video 1, where over 300 astrocytes are shown in a cleared section of the CA1 part of a mouse hippocampus. Figure 4 presents the cube described in Video 2, where both astrocytes and excitatory neuronal somata are shown in thick transparent tissue. Figure 5 presents an entire hemisphere, as described in Video 3. Brain-wide neuronal projections are displayed from the dorsal CA1 (green, GFP) and the ventral CA1 (red, TdTomato).

Video 1 presents a thick image of hippocampal astrocytes (>300) acquired with a two-photon microscope after a CLARITY procedure. Fluorescence was provided by viral vector transduction specific to astrocytes. Video 2 presents the spatial proximity between astrocytes and the nuclei of hippocampal pyramidal cells. Video 3 traces axonal bundles across an entire hemisphere; axons project from the primary motor cortex and are traced back from the spinal cord. Another bundle is traced from the dorsal hippocampus toward the Supra Mammillary bodies. Lastly, it traces a red bundle of axons from the Mammillary bodies toward their origin at the ventral hippocampus. All marked cells are exclusively excitatory.

Figure 1
Figure 1: Detailed description of the clearing process. (A) Validation of fluorophore expression. In this example, all astrocytes in the CA1 part of a mouse hippocampus express a red protein (TdTomato, imaged in magenta), and all excitatory neurons express a green protein in their nuclei (H2B-GFP). This image was acquired using a 2-photon microscope. The image was taken using a water immersion 16x objective (0.8 NA) with magnification of 2.4 to obtain a 652 x 483 µm field of view, at 15.5 frames/s and a z-stack with 0.937 µm intervals between the planes. The specificity and penetrance were verified by immunohistochemistry9. (B) Transferring the N2 gas from the tank into the tube holding the brain requires a pipe with a needle at its end. (C) Air inside the tube is replaced with N2 by creating two holes, one (upper arrow) for the inflow of N2via the needle connected to the piping from the gas tank and another (bottom arrow) for outflow. (D) Covering the holes with modeling clay immediately after degassing prevents the N2 from exiting the tube. (E) Heating the degassed solution for 3.5 h in a warm bath. (F) Successful polymerization results in a brain embedded in gelled HS. Gas bubbles might be trapped in the polymer. (G) Immediately after extraction from the gel is the best point for the brain to be cut into the desired thickness. (H) Perforated tubes allow the flow of the solution on the stirrer to reach the sample. (I) Brains inside perforated tubes are held vertically by a self-made holder of a size made to fit a 2 L beaker, standing on a hotplate stirrer. (J) Example of a brain that is not clear enough. The sample should be stirred in the CS2 for approximately one more week. (K) Example of a sufficiently clear brain. (L) When inserting the clear brain into PBST, the tissue becomes whiter than before. This change of color is due to the Triton and will disappear when moved from this solution to the next. (M) After >24 h in the RIMS, the brain will become fully transparent again. Abbreviations: GFP = green fluorescent protein; NA = numerical aperture; HS = hydrogel solution; CS2 = Clearing Solution 2; PBST = Phosphate-buffered saline containing 0.5% Triton X-100; RIMS = Refractive Index Matching Solution. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Chamber preparation. (A) The first layer of hot glue walls covers the edges of a slide and leaves a space wide enough to contain the brain without touching the walls. One hole is left in the upper-left corner of the slide. (B) Adding layer after layer to reach the height of the tissue. (C) Straight coverslip (white arrow) seals the chamber. (D) Chamber filled with RIMS through the hole in the chamber walls. (E) The chamber is filled with RIMS leaving no air bubbles. (F) Sealing the hole in the chamber with hot glue to prevent any leakage. Side view. (G) At this stage, any hot glue remnant extending over the edges of the slide should be cut off the chamber. (H) Fully prepared chamber, ready for imaging under the microscope. Abbreviation: RIMS = Refractive Index Matching Solution. Please click here to view a larger version of this figure.

Figure 3
Figure 3: All imaged astrocytes (AAV1-GFAP::TdTomato, red) in a cleared hippocampus. A single frame from Video 1 capturing all imaged astrocytes (AAV1-GFAP::TdTomato, red) in a cleared hippocampus. All details of the image properties are described under Video 1. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Astrocytes (AAV1-GFAP::TdTomato, red) and hippocampal pyramidal cells nuclei (AAV5-CaMKII::H2B-eGFP, green) in thick transparent tissue. A single frame from Video 2 showing both astrocytes (AAV1-GFAP::TdTomato, red) and hippocampal pyramidal cells nuclei (AAV5-CaMKII::H2B-eGFP, green) in thick transparent tissue. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Green axons (AAV5-CaMKII::eGFP, green) and red axons (AAV5-CaMKII::TdTomato). A single frame from Video 3 showing both types of axons derived from several locations in a mouse brain that can be traced throughout an entire hemisphere. Please click here to view a larger version of this figure.

Video 1: Footage of >1 mm thick slice of hippocampal dorsal CA1 astrocytes (AAV1-GFAP::TdTomato, red). The video was acquired with a two-photon microscope using a 16x objective (0.8 NA) with magnification of 2.4 following a CLARITY procedure. Similar to Figure 1A, the z-stack interval was set to 0.937 µm. Fluorescence was provided by viral vector transduction specific to astrocytes. The number of astrocytes in this cube is >300, and most processes are available for analysis in most cells. Abbreviations: NA = numerical aperture; AAV = adeno-associated virus; GFAP = glial fibrillary acidic protein. Please click here to download this Video.

Video 2: Astrocytes and excitatory neurons nuclei in a 3D cube. The cube contains all lamina of the dCA1 under the optical conditions mentioned for Figure 1A and Video 1. This cube exhibits the spatial proximity between astrocytes (AAV1-GFAP::TdTomato, red) and the nuclei of hippocampal pyramidal cells (AAV5-CaMKII::H2B-eGFP, green). Abbreviations: AAV = adeno-associated virus; GFAP = glial fibrillary acidic protein; CAMKII = calcium/calmodulin-dependent protein kinase II; eGFP = enhanced green fluorescent protein; H2B = histone 2B. Please click here to download this Video.

Video 3: Tracing axonal bundles across an entire hemisphere. Green axons (AAV5-CaMKII::eGFP, green) ending at the spinal cord are easily traced back to the primary motor cortex (more than 7 mm apart). From the hippocampus (dorsal side), another bundle is traced toward the Supra Mammillary bodies (most ventral side of the brain), a trace of almost 3 mm long. The second half of the video traces a red bundle of axons (AAV5-CaMKII::tdTomato) originating at the ventral hippocampus back from their target location at the Mammillary bodies. The image was taken under an Olympus scanning laser confocal microscope FV1000 using a 4x objective (0.16 NA), and multiple ROIs were combined using MATL FluoView-feature to present the entire hemisphere. Abbreviations: AAV = adeno-associated virus; CAMKII = calcium/calmodulin-dependent protein kinase II; eGFP = enhanced green fluorescent protein; NA = numerical aperture; MATL = multiple area time-lapse. Please click here to download this Video.

Hydrogel Solution (1 L)
Material Amount
VA-044 Initiator 2.5 g
PFA 4% 900 mL
Acrylamide (40%) 50 mL
Bisacrylamide (2%) 50 mL

Table 1: Preparation of hydrogel solution (1 L). Hydrogel solution must be prepared on an ice-cold surface. The order in which the ingredients are mixed is not important. Note that the total percentage of acrylamide is 2% unlike similar protocols that use either 1%17 or 4%22.

Clearing Solutions (1 L)
Material Amount
Boric acid (M.W. = 61.83 g/mol) 12.366 g 3.4 g
SDS 80 g 80 g
Tris base (M.W. = 121.14 g/mol) 0 12.1 g
Distilled Water (DW) Fill to 1 L Fill to 1 L
NaOH pH 8.5
1 L of Phosphate-buffered Saline with 0.5% Triton X-100
Material Amount
PBS 995 mL
Triton X-100 5 mL

Table 2: Preparation of clearing solutions 1 and 2 (1 L) and 1 L of Phosphate-buffered Saline with 0.5% Triton X-100. The clearing solutions can be prepared in advance and kept at room temperature for a long period. Any solution containing boric acid and SDS should be handled under a fume hood to avoid inhalation or skin contact with either SDS or boric acid. The NaOH in CS1 should be added last to set the pH at 8.5. Each brain sample should be placed in at least 5-25 mL of CS1 and kept overnight for sufficient diffusion. Abbreviations: CS1 = clearing solution 1; SDS = sodium dodecyl sulfate; M.W. = molecular weight.

Product Name Primary advantage Primary disadvantage
RapiClear Sample stays transparent Tissue swelling
RapiClear CS Sample shrinks back to normal size Sample may lose transparency

Table 3: RIMS options with advantages and disadvantages. Rapiclear (RC, RI = 1.47) or CLARITY-specific Rapiclear (CSRC, RI = 1.45). The primary difference between these two solutions is that while the CSRC shrinks the clear brain back to its original size, the RC solution does not, leaving the cleared sample slightly swollen. Abbreviation: RI = refractive index.

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Tissue clearing methods present a revolutionary tool in brain research, inviting questions that could not previously have been asked. From targeting the properties of a small group of cells, a single cell, or even a single synapse, CLARITY now enables the targeting of total cell populations or long-range connectivity features by using relevant fluorophores.

The outcome of the fluorophore expression and CLARITY procedure combination is not binary; many factors may interfere with the procedure leading to suboptimal results. First, the fluorophore expression must be verified in advance. As the CLARITY procedure causes some information loss, sufficient fluorophore protein expression is crucial to the validity of the image at the end of the CLARITY process. Second, the transcranial perfusion must be handled carefully, ensuring that all blood exits the brain, because the autofluorescence of blood will lead to unreliable data in thick slices. Third, all parameters discussed in the protocol must be handled in each step of the clearing process with precision, e.g., O2 residue in the HS before polymerization or temperature imprecision at each stage will prevent the chemical reaction between the active materials from occurring.

Previous protocols17,22 recommend different concentrations of acrylamide in the HS. The primary purpose of acrylamide is to better fixate the molecules with amine residues (proteins and nucleic acids). Slight alterations to the concentration greatly impact the entire clearing process: if the fixation is too compact, the lipid molecules will not disconnect from the tissue, and the clearing process will be unnecessarily protracted or result in opaque brains. Lower concentrations of acrylamide, however, will not adequately maintain the brain structure once devoid of lipids. In this protocol, the acrylamide percentage is set to provide the delicate balance of fixated yet clear tissue.

Most of the clearing process takes place in CS2, and the tissue clarity level should be tested daily. PBST cleanup is an essential step between the Clearing solutions and the refractive index matching solutions because it cleans the residual SDS molecules, which may interact with the RIMS. It is also possible to keep the clear tissue in PBST (0.5%) indefinitely if chamber preparation is not yet needed.

When placing the brain in the RIMS, one should become familiar with the benefits and limitations of the solutions. For example, RapiClear will cause complete transparency even in almost-cleared tissues, but will also cause some swelling, which should not be neglected when analyzing the data. Previous measurements9 suggest equal expansion along all axes (i.e., dorsoventral, anterior-posterior, and lateral-medial axes), making it possible to calculate the index of swelling by comparison to thin slices from the same brain region. Using CLARITY-Specific RapiClear eliminates the swelling; however, if any SDS leftovers remain in the tissue, they will aggregate into non-transparent masses.

Another benefit of this modified protocol is its cost. Previous protocols suggest different glue types to create a chamber that can hold the brain in RIMS. In this protocol, we simply use hot glue. It does not react with the solutions, is available to purchase everywhere, is easy to use, and is significantly cheaper than the glues suggested before.

Data acquired from the imaging of thick brain samples can describe entire cell populations, connectivity across brain structures, or the tracking of a single axon across large distances (Video 3). Although questions regarding these characteristics have been asked before, the validity of the data now achievable using thick brain slices greatly improves upon prior, mistake-prone methods of thin-slice image overlay.

The CLARITY process grants successful and uniform clearing of thick slices-from several millimeters to full brain imaging. The passive clearing (i.e., not using ETC) diminishes the risk of harm to the tissue, and the time for which samples are immersed in clearing solutions so that it does not affect the high efficiency of protein conservation.

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The authors have no conflicts of interest to disclose.


This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 803589), the Israel Science Foundation (ISF grant No. 1815/18), and the Canada-Israel grants (CIHR-ISF, grant No. 2591/18). We thank Nechama Novick for commenting on the whole manuscript.


Name Company Catalog Number Comments
AAV1-GFAP::TdTomato ELSC Vector Core Facility (EVCF) viral vector used to detect astrocytes
AAV5-CaMKII::eGFP ELSC Vector Core Facility (EVCF) viral vector used to detect neurons
AAV5-CaMKII::H2B-eGFP ELSC Vector Core Facility (EVCF) viral vector used to detect neuronal nuclei
AAV5-CaMKII::TdTomato ELSC Vector Core Facility (EVCF) viral vector used to detect neurons
Acrylamide (40%) Bio-rad #161-0140
Bisacrylamide (2%) Bio-rad #161-0142
Boric acid Sigma #B7901 Molecular weight - 61.83 g/mol
Confocal microscope, scanning, FV1000 Olympus 4x objective (UPlanSApo, 0.16 NA)
Imaris software Bitplane, UK A software that allows 3D analysis of images
NaOH Sigma #S5881
PFA 4% EMS #15710
RapiClear SunJin lab #RC147002
RapiClear CS SunJin lab #RCCS002
SDS Sigma #L3771
SyGlass software A software that allows 3D analysis of images using virtual reality
Tris base 1 M Bio-rad #002009239100 Molecular weight - 121.14 g/mol
Triton X-100 ChemCruz #sc-29112A
Two photon microscope Neurolabware Ti:sapphire laser (Chameleon Discovery TPC, Coherent), GaAsP photo-multiplier tubes (Hamamatsu, H10770-40) , bandpass filter (Semrock), water immersion 16x objective (Nikon, 0.8 NA) 
VA-044 Initiator Wako #011-19365



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Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains
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Refaeli, R., Goshen, I.More

Refaeli, R., Goshen, I. Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains. J. Vis. Exp. (181), e63679, doi:10.3791/63679 (2022).

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