Sep 18, 2025
Whole-brain Staining and Tissue Clearing Protocol of LINCS
- Shilin Zhong1,
- Minmin Luo2,3,4,5,
- Rui Lin1,6
- 1National Institute of Biological Sciences (NIBS), Beijing, China;
- 2Chinese Institute for Brain Research (CIBR), Beijing, China;
- 3New Cornerstone Science Laboratory, Shenzhen, China;
- 4Research Unit of Medical Neurobiology, Chinese Academy of Medical Sciences, Beijing, China;
- 5Beijing Institute for Brain Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China;
- 6Tsinghua Institute of Multidisciplinary Biomedical Research (TIMBR), Beijing, China.
Protocol Citation: Shilin Zhong, Minmin Luo, Rui Lin 2025. Whole-brain Staining and Tissue Clearing Protocol of LINCS. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwmokv8j/v1
Manuscript citation:
Zhong et al., Ultrabright chemical labeling enables rapid neural connectivity profiling in large tissue samples, Neuron (2025), https://doi.org/10.1016/j.neuron.2025.08.022
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: November 03, 2024
Last Modified: September 18, 2025
Protocol Integer ID: 111456
Keywords: LINCS, ultrabright neuronal labeling system, tissue clearing protocol of linc, lincs with tissue clearing, brain staining, tissue clearing protocol, tissue clearing, sheet microscopy, neuron, type specific labeling with chemical dye, linc, labeling, specific labeling, chemical dye, mount staining procedure, integrating linc, cell
Abstract
Here we present LINCS (Labeling Individual Neurons with Chemical dyes and controllable Sparseness), an ultrabright neuronal labeling system that achieves cell-type specific labeling with chemical dyes. We established and optimzed an efficient pipeline for rapid whole-mount staining procedure, integrating LINCS with tissue clearing and commercially available light-sheet microscopy.
Image Attribution
Created in BioRender. LIN, R. (2025) https://BioRender.com/giul03e
Materials
Buffer
PTx.2
0.2% TritonX-100 in PBS
PTx.5
0.5% TritonX-100 in PBS
Reagent
Biotin (300 mM stock in DMSO stored at -20℃; completely dissolved before diluted in water; Sigma-Aldrich, V900418)
Heparin sodium (10 mg/mL stock)
NaN3 (10% solution)(can be substituted with ProClin)
20%, 40%, 60%, 80%, 100% Methanol (v/v in ddH2O)
Dichloromethane (DCM, J&K, 908525)
H2O2 (30% solution)
Alexa Fluor 647-conjugated monovalent Streptavidin (monovalent SA-AF647, Abcam, ab272190)
Dibenzyl ether (DBE, Sigma, 108014)
Troubleshooting
Safety warnings
Refer to the material safety data sheets (MSDS) for proper handling and usage guidelines for NaN3, DCM and DBE.
Ethics statement
Animal care and use followed the approval of the Animal Care and Use Committee of the National Institute of Biological Sciences, Beijing (Approval ID: NIBS2023M0013), in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals of China.
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Before start
- Tissue clearing is processed based on a modified iDISCO+ protocol.
- Steps 5-9,11,14-16 are done with gentle shaking on a rocker (~5 rpm).
- Steps 12-13 are done with gentle shaking on a shaker (~120 rpm).
- Perform all steps in closed tubes (Eppendorf 5 mL tubes), ensuring they are fully filled to prevent oxidation.
- It is recommended to cover marker labels on tube caps with transparent tape to prevent dissolution by organic reagents.
- Keeping the samples in dark after step 11.
- Samples should not be exposed to water after step 16.
Buffer
PTx.2
0.2% TritonX-100 in PBS
PTx.5
0.5% TritonX-100 in PBS
Reagent
Biotin (300 mM stock in DMSO stored at -20℃; completely dissolved before diluted in water; Sigma-Aldrich, V900418)
Heparin sodium (10 mg/mL stock)
NaN3 (10% solution; can be substituted with ProClin)
20%, 40%, 60%, 80%, 100% Methanol (v/v in ddH2O)
Dichloromethane (DCM, J&K, 908525)
H2O2 (30% solution)
Alexa Fluor 647-conjugated monovalent Streptavidin (monovalent SA-AF647, Abcam, ab272190)
Dibenzyl ether (DBE, Sigma, 108014)
LINCS workflow summary
Specific neuronal populations are labeled in vivo using seTurboID, delivered via AAV in Cre transgenic mice. Biotin is administered through drinking water. Three weeks after viral injection, brains are harvested and fixed in 4% paraformaldehyde (PFA). The tissue then undergoes pretreatment, whole-mount staining, and clearing procedures before light-sheet microscopy imaging.
AAV injection and biotin administration
AAVs is delivered to target regions at a rate of 23 or 46 nL/min using a Nanoliter 2020 Injector pump controlled by a Micro4 controller (WPI). Tissue samples are typically harvested 3 weeks post-injection to allow for sufficient transgene expression.
One week prior to sacrifice and perfusion, mice are administered biotin (2 mM) in their drinking water ad libitum. The biotin-supplemented water should be provided in sterilized bottles and replaced weekly.
Note: Extending the administration period beyond one week does not affect the final labeling efficiency.
Sample collection and fixation
1d
Anesthetize the mouse and perform intracardial perfusion, first with ice-cold heparinized PBS (10 U/mL concentration), then with ice-cold 4% PFA, followed by ice-cold heparinized PBS.
Harvest the brains and post-fix in 4% PFA o/n at 4℃ and then for 1 h at RT.
18h
Sample Pretreatment
2d
Wash post-fixed samples with PBS for 3x30 min at RT.
1h 30m
Dehydrate samples at RT by incubating them in a graded methanol series (20%, 40%, 60%, 80%, v/v in ddH2O, 1h for each), followed by two 1 h incubations in 100% methanol.
6h
Delipidate samples in 66% DCM (v/v in methanol) at RT o/n.
18h
Wash samples with 100% methanol for 2x2 h at RT.
4h
Pre-cool samples at 4℃ and then bleach samples in 5% H2O2 (v/v in ice-cold methanol) at 4℃ o/n.
18h
Rehydrate samples at RT by incubating them in a graded methanol series (80%, 60%, 40%, 20%, v/v in ddH2O, 1h for each), followed by two 1h incubations in PTx.2.
6h
Staining
4d
Stain samples with monovalent SA-AF647 (1:100 diluted for bulk labeling; 1:250 diluted for sparse labeling) in PTx.5 supplemented with 0.05% NaN3 for 3 days at 37–42℃ (37℃ is recommended for sparse labeling samples).
3d
Wash sampels with PTx.5 first for 2x1 h at RT, and then o/n at 25–37℃ (the lower temperature is recommended for samples with labeled axons enriched in cortical regions; Adding 0.05% NaN3 to PTx.5 is recommended for o/n washing).
14h
Wash samples with PBS for 4x1 h at RT.
4h
Tissue Clearing
1d
Dehydrate samples at RT by incubating them in a graded methanol series (20%, 40%, 60%, 80%, v/v in ddH2O, 1h for each), followed by two 1 h incubations in 100% methanol.
Note: Can stop optionally at this step o/n at RT.
6h
Delipidate samples in 66% DCM (v/v in methanol) for 3 h at RT. Wash samples for 2x15 min in 100% DCM.
3h 30m
Transfer samples to DBE in a new tube and store at RT until clear. The tube should be fully filled with DBE to prevent oxidation.
Before imaging, gently invert the tube to ensure the solution is homogeneous.
Note: It is recommended to perform imaging within one week.
Protocol references
Zhong et al., Ultrabright chemical labeling enables rapid neural connectivity profiling in large tissue samples, Neuron (2025)
Renieret et al., Mapping of brain activity by automated volume analysis of immediate early genes. Cell (2016)