Nov 04, 2025
SMART brain mapping
- Xiaowei Hu1,
- Yan Shen1,
- Yi Yang1,
- Guo-Qiang Bi1,
- Fang Xu1
- 1Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences
Protocol Citation: Xiaowei Hu, Yan Shen, Yi Yang, Guo-Qiang Bi, Fang Xu 2025. SMART brain mapping . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq658klk5/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: April 03, 2025
Last Modified: November 04, 2025
Protocol Integer ID: 126066
Keywords: smart brain mapping pipeline of marmoset brain, smart brain mapping pipeline, smart brain mapping, marmoset brain, brain reconstruction, throughput brain mapping, regulatory standards for animal experimentation, animal experimental procedure, animal experimentation, tissue clearing, brain, 3d microscopy, institutional animal care, experimental procedure
Funders Acknowledgements:
National Natural Science Foundation of China
Grant ID: 32000696
Guangdong Basic and Applied Basic Research Foundation
Grant ID: 2021A1515010625
Shenzhen Science and Technology Program
Grant ID: RCBS20200714114909001
STI 2030-Major Projects
Grant ID: 2022ZD0205203
Shenzhen Science and Technology Program
Grant ID: RCYX20210706092100003
Shenzhen Medical Research Funds grant
Grant ID: A2303005
Youth Innovation Promotion Association CAS grant
Grant ID: 2022367
Abstract
This protocol is for the Serial sectioning and clearing, 3D Microscopy, semi-Automated Reconstruction and Tracing (SMART) strategy for high-throughput brain mapping. The SMART brain mapping pipeline of marmoset brains includes viral labeling (optional), preprocessing (post-fixation), serial sectioning, tissue clearing and staining (optional), VISoR2 volumetric microscopy, semi-automated whole-brain reconstruction, and fiber tracing (optional).
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS), and conducted in an AAALAC-certified facility. Researchers applying this protocol should ensure compliance with local ethical and regulatory standards for animal experimentation.
Materials
Animal
Marmosets were individually housed under conditions that adhered to
institutional guidelines of CEBSIT, with a 12-hour light/dark cycle (light on
from 7 a.m. to 7 p.m.), a humidity and temperature-controlled environment (27°C
to 30°C), and ad libitum access to food and water. A 7.5-year-old male marmoset
was used in this protocol.
▲ CAUTION: The animal studies and procedures were approved (ION-2019011) by the
Animal Care and Use Committee of the Center for Excellence in Brain Science
& Intelligence Technology (CEBSIT), Chinese Academy of Sciences, Shanghai,
China.
Reagents
Reagents for surgery and viral labeling
l Anesthetics
for marmoset: Isoflurane (1.5% final concentration, delivered in 100% oxygen,
RWD, cat. no. R510-22-10) may be used.
▲ CAUTION Isoflurane
is a highly volatile substance and a proper device like anesthesia induction box or breathing mask
for animal should be used
l AAV-Cap-B10,
[ssAAV.hSyn.EGFP.WPREs.SV40pA]
Reagents for animal perfusion and tissue fixation
l All reagents
can be purchased from common vendors.
l Gibco 10×
phosphate-buffered saline (PBS), pH 7.4 (Fisher Scientific, cat.no. 70011044)
l Paraformaldehyde
(PFA), 8% (Solarbio Life Science, cat.no.P1112)
▲ CAUTION PFA is
toxic. Perform all procedures in a fume hood. Avoid inhalation and
exposure
to the skin or the eyes.
▲ CRITICAL Dilute 8%PFA to 4% final concentration to
prepare HMS solution described in “Reagent setup”.
l Acrylamide
(Aladdin, cat.no.A108465)
l N,N’-Methylenebisacrylamide
(Sigma, cat.no. M7279)
l 2,2'-Azobis
[2-(2-imidazolin-2-yl) propane] dihydrochloride (VA-044), (Wako, cat.no.
223-02112)
▲ CRITICAL VA-044 is a
heat -sensitive azo polymerization initiator, thus the powder or the aqueous
mixture should be stored at 4℃ until polymerization step.
Reagents for
tissue embedding
l Bovine serum
albumin ( Sigma, cat.no. V900933)
l Hydrogel
monomer solution (4%)
▲ CRITICAL
4% HMS can be prepared as described in ‘Reagent setup’.
Reagents for
smart brain mapping
l Triton X-100 (Sigma, cat. no. T928)
l Gibco 10×
phosphate-buffered saline (PBS), pH 7.4 (Fisher Scientific, cat.no. 70011044)
l PC-300
(Solarbio, cat.no. P6840)
▲ CRITICAL PC300 is
used here as a preservative.
l Agarose, low
melting (Sangon, cat.no. A600015-0025)
l 4',
6-diamidino-2-phenylindole (DAPI, Beyotime
Biotechnology, cat. no. C1006)
l NeuroTrace‱
640/660 deep red fluorescent Nissl stain (NT640, ThermoFisher, cat. no. N21483)
l Iohexol (
Hisyn Pharmaceutical, cat. no. 29242990.99 )
l Urea (
Sangon, cat. no. A600148-0002)
l 2,2’,2”,-nitrilotriethanol
( Sigma, cat.no. V900257)
l Sodium
chloride (Sigma, cat. no. S5886)
l Potassium
chloride (Macklin, cat. no. C16403322)
l Sodium
phosphate dibasic (Sigma, cat.no. S5136)
l Sodium
bicarbonate (Sigma, cat. no. S5761)
Equipment
l 50 ml conical tube (Corning, cat. no. 352070 or Greiner, cat. no. 227261)
l 24-gaugue catheter (Introcan-w,
cat.no. 4254074B)
l 2ml Sterilized Microcentrifuge Tubes, enzyme-free(Keewin, cat.no.
A211-CT02002)
l Inhalation Anesthesia Machine (RWD, cat.no.R530)
l Oscillating Incubator(Shanghai Zhichu Instrument, cat.no.ZQTY-90E)
l Oscillating Incubator(Shanghai Zhichu Instrument, cat.no.ZQZY-A8)
l BOD Cooling Incubators（Shanghai Zhicheng, cat.no.ZXMP-R1230）
l Circular Decolorization Shaker(Dlab, cat.no.SK-0180-S)
l Vortex mixer( Ohaus, cat.no.VXMNAL)
l Ice maker( Xueke, cat.no. IMS-60)
l Tissue culture plate, 6 well(Falcon, cat.no. 353046)
l Screw-thread Wide-mouth Bottle, 125ml(CNM, cat.no.SGEQ-2180125)
l Vacuum desiccator(Shanghai Yueci, cat.no. PC-3)
l Vibroslicer (Precisionary Instruments, cat.no.Compresstome VF-800)
l VISoR2 imaging systems ( Bitelligen, cat.no.VISoR M1)
l 9.4T/12-cm MRI( Bruker Biospin)
l Reagent bottle 1000ml
l Chinese brush
l Mounting glass
l Coverlip
l Patching chamber
l Imaging chamber
l Digital Display Refractometer(ATAGO, cat.no.PAL-RI)
l Microwave
oven
l Fume hood
l Heating blanket
Data analyze tools
l VISoR Reconstruction
(https://github.com/SMART-pipeline/Volume-reconstruction)
l
Reagent setup
1×PBS, 20L stock
solution In ddH2O add 160gNaCl, 4g KCl, 28.4g Na2HPO4,
5.4g KH2PO4. Stir until dissolved and the solution is stable at room
temperature for 2 months.
40% (w/v) acrylamide
solution Prepare 16g of acrylamide and then add distilled
water to 40ml. Shake until fully dissolved and then store at 4℃ for up to 1
month.
2% (w/v) bisacrylamide
solution Prepare 0.8g of N,N’-Methylenebisacrylamide and
then add distilled water to 40ml. Shake until fully dissolved and then store at
4℃ for up to 1 month.
4% HMS 40ml 4% Hydrogel monomer solution (HMS, 4%) which was prepared for
perfusion by mixing 4ml 40% acrylamide (4% final concentration), 1ml 2%
bisacrylamide (0.05% final concentration), 4ml 10×phosphate buffered saline (PBS) (1×final concentration), 20ml 8% (w/v)
paraformaldehyde (4% final concentration), 11ml distilled water and 0.5g VA-044
thermal initiator (1.25% final concentration). The preparation process should
be carried out on ice and in fume hood. The final mixture can be stored at 4℃
for 1-2 week.
▲ CRITICAL: the mixture could polymerize as hydrogel when the
temperature rises.
20%BSA Dilute
8g of Bovine serum albumin powder in distilled water to final volume 40ml.
▲ CRITICAL: the solution will generate foam after shaking. It
is necessary to wait until the foam completely dissipates before conducting the
volume-fixing. The solution should be prepared before the day of experiment and
keep in 4℃ for no more than 1 week.
4%Agrose Dilute
1.6g Agarose powder in ddH2O to 40ml. The agarose would melt through
heating in a microwave oven.
5% TritonX-100 in PBS
(PBST) Dilute 5ml Triton X-100, 10ml 10×phosphate buffered
saline and 0.1ml PC-300 into 84.9ml distilled water. The solution keep stable
for 1 month.
Preparation of slice
staining solution On the day of
experiment, mix 40ml fluorescent dyeing
solution containing 35.36 ml 4′,6-diamidino-2-phenylindole (DAPI) stock
solution (79.4% final concentration) for nucleus , 0.4ml NeuroTrace 640/660
deep-red fluorescent Nissl stain (NT640, 1% final concentration), 0.2ml Triton X-100 (0.5% final
concentration), 0.04ml PC-300( 0.1% final concentration), 4ml 10×PBS（1×final
concentration)
▲ CRITICAL: the final concerntration of NeuroTrace 640/660
deep-red fluorescent Nissl stain should vary depend on the sample relating to
the slice thicknenss and tissue density.
PuClear refractive
index matching solution The RI matching solution is prepared by mixing 650g Iohexol (50 wt%) , 300g urea (23wt%), 140g 2,2’,2”,-nitrilotriethanol (11wt%) and 210ml distilled water(16wt%). Then place the reagent bottol into Oscillating Incubator(Shanghai Zhichu Instrument, cat.no.ZQZY-A8) shaking for 48h at 180rpm in 37℃. Test the final RI to 1.52 using Digital Display Refractometer(ATAGO, cat.no.PAL-RI). The matching solution keep stable in 1 month.
Troubleshooting
Whole-brain Viral labeling (Optional)
For Marmoset
Note
CAUTION: An appropriate viral labeling method could be altered according to certain experimental design.
Implement fasting and water restriction for at least 6 h before surgery.
Note
Caution: Fasting and water restriction are required to prevent gastric regurgitation.
Anaesthetize the marmoset with 1.5% -3% isoflurane inhalation in oxygen before surgery.
Note
Critical: The anesthetic concentration should be adjusted per the animal's condition intraoperatively.
Shave the skin over the tail vein and sanitize with an ethanol scrub.
Inject of 2 mL of the tracer (AAV-Cap-B10, [ssAAV.hSyn.EGFP.WPREs.SV40pA], 1 × 1013 gc/mL) through a 24-gauge catheter by tail vein injection over several minutes.
Recover the animal on a warm blanket (37 °C) until they regained normal motor functions.
Set the animal back to cages and monitor closely for normal behavior over the 4d, followed by daily
obervations thereafter.
Euthanize the marmoset for brain harvest 8 weeks after injection.
For Mouse
Anaesthetize the mice with 1.5%-2.0% isoflurane inhalation in oxygen before surgery.
Inject 100μl of the same crossing BBB viral labeling tracer as used on marmoset(AAV-Cap-B10, [ssAAV.hSyn.EGFP.WPREs.SV40pA], 1 × 1013 gc/mL, 1:10000 dilution) through 24-gauge catheter by retro-orbital vein injection over several minutes.
Note
CAUTION: If bleeding occurs after injection, conduct hemostasis and anti-inflammation treatment in time and make a record. The injection volume may be affected by bleeding.
Recover the animal on a warm blanket (37 °C) until they regained normal motor functions.
Euthanize the mice for brain harvest 8 weeks after injection.
Specific circuit labeling (Optional)
For Marmoset
Anaesthetize the marmoset with 1.5-3.0%(v/v) isoflurane inhalation in oxygen by placing the animal in an induction box before surgery.
Connect the animal to the bench mask with about 1.5% -3.0% isoflurane-oxygen gas flow, using a stereotaxic apparatus to fix the head of the marmoset. The optional step is performing oral tracheal intubation, then connecting the gas anesthesia pipeline and equipment.
Place the temperature probe into the anus or between the belly and the blanket, check the temperature, heart rate and SpO2 during the process.
Screw the stereotaxic frame to the base plate with the anterior/posterior (A/P) rail and medial/ lateral (M/L) rail.
Note
CRITICAL: Avoid head movement during surgical procedures to reach the accurate determination of stereotaxic coordinates.
Shave the skin with washes of povidone iodine, followed by 75% ethanol.
Incise the skin over the sagittal midline with a sterile surgical blade.
Note
CRITICAL: Surgical instruments and procedures must be sterile.
Check the position of the microsyringe orthogonal to the x/y/z axes, and place the skull in a horizontal position.
Measure the injection sites according to brain atlas or MRI result; mark the borders around the craniotomy: the occipital region (ventral visual area 4) of the left hemisphere (AP, -3.79mm; ML, 6.79mm) with a sterile pen.
Note
CAUTION: When using the anatomical atlas for stereotaxic injections, actual injection coordinates may deviate due to individual biological variations in brain morphology, size, and landmark positioning.
Perform a craniotomy at the marked position with a dental drill and keep the bone fragment in sterile saline buffer.
Note
CRITICAL: If bleeding occurs after injection, conduct hemostasis and anti-inflammation treatment in time. Avoid puncturing the dura mater during this procedure.
Make sure the injection needle is not blocked if it touches blood.
Gently rinse the drilled area with sterile saline buffer, and employ the microsyringe injector which is filled with 1000 nL rAAV mixture (AAV2/9, [rAAV-hSyn-SV40-NLS-Cre] 1/10000, [rAAV-CAG-DIO-tdTomato] 1/4) to administer the virus gradually into the depth within the target brain region mentioned in step 3.8 (AP, -3.79mm; ML, 6.79mm; DV, 9.75mm). The injection is performed at rate of 200 nL/min and maintain above 20 minutes (including 5 min both following insertion and before withdrawal of the microsyringe).
Note
CRITICAL: Make sure the injection needle is not blocked if it touches blood.
Place the bone fragment back in its original position and apply some bone wax around the borders.
Suture the incision. Administer the antibiotics to the animal for 3 days after surgery.
Return the animal to the home cage after it is fully alert. Monitor the animal for a further 1–2 h in the home cage and twice a day until total recovery
For Macaque
For macaques, the stereotaxic injection process could refer to the above method for marmosets.
Note
CRITICAL: Injection sites could be further confirmed from reconstructed whole-brain
images
Brain brain acquisition
For Marmoset
Transcardial perfuse the marmoset after aneasthetized with 1.5% isoflurane.
Infuse 500mL PBS at speed 30mL/min in 16min, and 100mL 4% HMS at rate of 40mL/min in 2.5min, 250mL 4%HMS (30mL/min) in 8min, 1L 4% HMS(20mL/min) 50min.
Note
CRITICAL: All the solution should be stored and operated in 4℃.
Extract the brain immediately after perfusion, within 30 min.
For Macaque
Anesthetized macaques by 10 mg/kg ketamine hydrochloride injection (i.m.,50 mg/mL) and maintained with 20 mg/kg pentobarbital sodium (i.m.,40 mg/mL)
Perform transcardial perfusions thirty minutes after anesthesia, by sequentially perfusing with the following solutions at the specified speed: PBS 8 L (37°C, 10 mL/s), PBS 1 L (4°C, 1.5 mL/s), 4% HMS 1L (4°C, 1.5 mL/s), and 4% HMS 1 L (4°C, 0.3 mL/s)
Extract the brain immediately after perfusion
Mouse( Including vasculature labeling)
After anesthetizing the mice via inhalation with 1.5% isoflurane in oxygen, inject 75 μL of DyLight 488- or DyLight 649-conjugated Lycopersicon esculentum lectin via retro-orbital route.
Note
CAUTION: This is a optional step according to the experimental requirements. The labeling reagent can be replaced as per the experimental.
One hour post-injection, administer secondary anesthesia via intraperitoneal injection of a ketamine/xylazine mixture (dose:10 mg/kg ketamine hydrochloride; 1 mg/kg xylazine hydrochloride).
Perfuse the follow solutions in sequence: 30 mL of PBS (37 °C, 10ml/ min), 30 mL of PBS (4 °C , 10ml/ min), 30 mL of 4% PFA (4°C, 10ml/ min), and 75 μL of lectin dissolved in 30 mL of ice-cold PBS (4°C, 3ml/ min).
Note
CRITICAL: Remove the lectin solution when referring to this procedure for perfusion without the requirement of labeling vasculature
Extract the brain immediately after perfusion and store temporarily in 4% PFA.
Post-fixation and embedding
For standard samples
Immerse the brain sample in 4%HMS and store at 4 °C for 2–7 days, depending on brain size and tissue density (typically 2 days for mouse brains; 7 days for primate brains)
Transfer the brain sample to the embedding solution, a 1:1 mixture of 4% HMS (2% final concentration) and 20% bovine serum albumin (10% final concentration) and incubate in the sulution for for 0.5–7 days in 4℃, again depending on tissue size and density
Transfer the brain and embedding solution to Constant Temperature & Humidity Incubator, polymerize at 37℃ for 4-5 h.
Wash 3 times in PBS for 1 h each time to remove residual reagents.
For immunolabeling
Note
CRITICAL: These alterations have the potential to boost the efficiency of antibody penetration.
Post-fix the brain for 1-7days in 4% PFA instead of 4%HMS.
Note
CRITICAL: The time of post-fix is typically 1 day for mouse brains, 4 days for monkey brains and 7days or longer for human brains.
Wash the brains with PBS three times over a 24-hour period at RT to remove the PFA.
Immerse the brains in 10% fish skin gelatin solution(37℃，liquid) for 30minutes (the larger brain could extend the immersion time to 4 hours)
Note
CAUTION: The gelatin solution is in a solid state at room temperature. When using it, preheat it to 37°C in advance to restore it to a liquid state.
Locate the brain and 10% fishskin gelatin solution to a mold box of appropriate size and wait for the gelatin to set.
Place the mold containing the brain at 4°C for more than 4 hours.
Demold, trim the embedded material to a suitable size and make corner cuts for identifying the orientation.
Immerse the embedded brain in 4% PFA to solidify the gelatin for more than 20 hours.
Note
CRITICAL: Gelatin fixed by PFA can remain in a solid state at 37°C or higher temperatures.
Sectioning and clearing
Serial sectioning
Trim the embedding block and and mark a notch on the left ventral side to preserve orientation for whole-brain reconstruction.
Stick it onto the stage of vibroslicer with glue, fill the gap space with melting 4% agarose and wait for the agrost solidify.
Note
CRITICAL: Select a suitable vibratome according to the size of the samples.
Section the embedded brain into 300-µm-thick slices from the anterior segment of the coronal plane.
Note
CRITICAL: The thickness of sections can be altered (normally reduced to 150μm or 100μm) to achieve better staining effects according to the molecular sizes of dyes or antibodies.
Utilize a soft Chinese brush to gently lift out the brain slices. Mark the brain slices in a squential counting.
Place the brain slice into three 6-well cell culture plates, 3-5 with known serial numbers are placed in one well filled with PBS
Note
CRITICAL: For brain samples: use 12-well plates for mouse brains, 6-well plates for marmoset brains, and individual petri dishes for monkey and human brains.
Brain slices should be arranged cyclically according to the count per cycle, with clear distinguishability of slices within the same well.
Expected result
- Marmoset brains would be yielding approximately 70-105 slices per brain.
- Whole macaque brains generate approximately 200–250 slices per brain.
- Mouse brains yield approximately 40–50 slices per sample.
Store the plates at 4℃
Tissue Clearing
Treat the brain slices with a high concentration of Triton X-100 solution (5% in PBS) for1- 4 days at 37℃ in Oscillating Incubator at rate of 60rpm.
Note
CRITICAL: The clearing time for mouse brains is 1 day, while that for monkey and human brains is 4 days.
Staining
For general staining
Stain the brain slice with slice staing solutin for DNA and nissl stain for 2-4 days.
Note
CRITICAL: The staining time for mouse brains is 2 days, while that for monkey and human brains is 4 days.
Wash 4 times with PBS after stain.
For immunolabeling
Block the brain slices in 0.5M Glycine in 0.3%PBST for 4 hours at 37℃.
Note
CRITICAL: The BSA or serum used in conventional blocking may quench Nissl fluorescent dyes, glycine is employed here to block the nonspecific effects of aldehyde groups.
Wash 1 hour in PBS at RT.
Incubate the brain slices in primary antibodies dissolved in 0.3%PBST for 3-7days at 4℃.
Note
CRITICAL: The incubating time for mouse brains is 3 day, while that for monkey and human brains is 4-7 days.
Note
CRITICAL: When co-staining with different antibodies, pay attention to distinguishing the host species.
Wash 4 times with 0.3%PBST, 1-2 hours each time.
Perform the general staining if needed.
Note
CAUTION: General staining can be performed simultaneously with the secondary antibody, ensuring accurate final concentrations of all dyes.
Mounting and RI matching
Mounting
Trim off the excessive embedding, and remain the notch of slices.
Immerse the brain slice into 4%HMS, and transfer the slices on the hydrophilic mounting glass with the Chinese brush setting the left ventral side of each brain slice in one direction.
Stick suitable number in order and clip the mounting glass in the patching chamber
Drop an excessive amount of vacuum-treated HMS onto the brain slices, overlay a hydrophobic cover glass on the top
Fix the chamber and place it into a sealed box with few ddH2O below the chamber.
Put the box to Constant Temperature & Humidity Incubators at 37℃ for 3-4h.
Remove the cover glass and place the mounting glass with brain slices into PBS.
Refractive index matching
Fix the mounting glass into imaging chamber and fill the PuClear RI matching solution.
Incubate in the brain slices overnight to facilitate optical transparency.
VISoR2 Microscopy
10X/NA0.3 objectives equipped in these systems are used to generate raw images of the slices at 1×1×2.5 μm3 pixel resolution.
Triple-channel imaging of a marmoset brain generates 20 terabytes (TB) images after compression, with an approximate compression ratio of 8:1.
Dual-channel imaging of a rhesus macaque brain generates 500 TB raw images.
Four-channel imaging of a mouse brain generates 1 TB raw images.
Whole-brain reconstruction
Volume stitching of VISoR2 raw images are performed with custom codes (https://github.com/SMART-pipeline/Volume-reconstruction).
Semi-automated tracing (Optional)
The single axon reconstruction of the marmoset brain (CJ004) is semi-automatedly performed with the NeuroFly (beanli161514/neurofly: Some tools for neuronal image analysis) software.
Install Neurofly following the repository in Github: beanli161514/neurofly: Some tools for neuronal image analysis
Protocol references
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