Aug 13, 2025
Clearing of deparaffinized human brain tissue
- Danila Di Meo1,
- Josephine Ramazzotti1,
- Aron Emmi2,
- Irene Costantini1
- 1European Laboratory for Non Linear Spectroscopy (LENS), University of Florence, Italy;
- 2Institute of Human Anatomy, Department of Neuroscience, University of Padova, Italy.
- LENS
External link: https://www.biorxiv.org/content/10.1101/2024.09.10.612232v1
Protocol Citation: Danila Di Meo, Josephine Ramazzotti, Aron Emmi, Irene Costantini 2025. Clearing of deparaffinized human brain tissue . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3qmjev25/v1
Manuscript citation:
Danila Di Meo, Michele Sorelli, Josephine Ramazzotti, Franco Cheli, Samuel Bradley, Laura Perego, Beatrice Lorenzon, Giacomo Mazzamuto, Aron Emmi, Andrea Porzionato, Raffaele De Caro, Rita Garbelli, Dalila Biancheri, Cristiana Pelorosso, Valerio Conti, Renzo Guerrini, Francesco S. Pavone, Irene Costantini
"Quantitative cytoarchitectural phenotyping of deparaffinized human brain tissues"
bioRxiv 2024.09.10.612232; doi: https://doi.org/10.1101/2024.09.10.612232
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: March 04, 2025
Last Modified: August 13, 2025
Protocol Integer ID: 123768
Keywords: clearing , paraffin, deparaffinization, labelling, SHORT, DISCO, deparaffinized human brain tissue, human brain tissue applicable to different clearing protocol, human brain tissue, detailed version of the deparaffinization protocol, optical clearing, deparaffinization protocol, fluorescence microscopy technique, 3d reconstruction of the tissue, using fluorescence, microscopy technique, fluorescent labelling, different clearing protocol, clearing, tissue, 3d reconstruction
Funders Acknowledgements:
CONNECT
Grant ID: U01 NS132181
Abstract
This protocol is a detailed version of the deparaffinization protocol for human brain tissue applicable to different clearing protocols (SHORT and iDISCO). The method was optimized by the European Laboratory for Non-Linear Spectroscopy - University of Florence -Italy, and describes the process to deparaffinize human brain tissue to prepare them for optical clearing and fluorescent labelling, permitting the 3D reconstruction of the tissue at micrometer-resolution using fluorescence microscopy techniques.
Attachments
final_draft.pdf
2.9MB
Troubleshooting
Deparaffinization of brain tissue
Deparaffinization was achieved by first removing as much paraffin surrounding the tissue block as possible with a blade. To completely remove the remaining paraffin, samples were placed in a water bath at 60°C for 30-60 min until the paraffin was macroscopically eliminated. In order to speed up this step and completely clean samples from the paraffin, tissue blocks were positioned on a porous membrane inside a 50 ml tube floating in the water bath. Following the paraffin melting, tissue blocks were incubated 6-8 times in 98% Xylene for 30 minutes at RT, using a rotary shaker. At this point, tissue blocks should appear translucent. Afterwards, samples were washed with 100%, 95%, and 70% ethanol diluted with water at RT for 30 min respectively, and further washed 3 times with 1× PBS for 10 min at RT. After embedding in 4% agarose, tissue sections of 500 ± 50 μm-thick were obtained using a vibratome (Leica VT1000 S) and stored at 4°C in PBS + 0.01% w/v NaN3.
Application to SHORT tissue transformation method
Single deparaffinized slices with a thickness of 500 μm were processed with the SHORT tissue transformation method, as previously described. The specimens were first incubated in a Switch-Off solution (50% v/v 1× PBS (pH 3), 25% v/v 0.1 M hydrochloric acid (HCl), 25% v/v 0.1 M potassium hydrogen phthalate (KHP), and fresh 4% v/v glutaraldehyde) at 4°C in gentle shaking for 24 h. The solution was replaced with the Switch-On (1× PBS (pH 7.4) and fresh 1% v/v glutaraldehyde) and the incubation was performed at 4°C in gentle shaking for 24h. Afterwards, slices were washed 3 times for 2 h each in 1× PBS at RT and reactive glutaraldehyde was inactivated by o/n (overnight) incubation with an inactivation solution (1× PBS (pH 7.4), 4% w/v acetamide, 4% w/v glycine, pH 9.0) at 37°C in a water bath. Following inactivation, the samples were washed in PBS at RT and then incubated in the clearing solution (200 mM sodium dodecyl sulfate (SDS), 20 mM sodium sulfite (Na2SO3), and 20 mM boric acid (H3BO3), pH 9.0) for 3-6 days at 55°C. After the clearing step, samples were extensively washed in 1× PBS + 0.1% Triton X-100 (PBST) at 37°C for 24 h. Next, the slices were treated with 30% H2O2 for 45 min and antigens were unmasked with the preheated antigen retrieval solution (10 mM Tris base (v/v), 1 mM EDTA solution (w/v), 0.05% Tween 20 (v/v), pH 9) for 10 min at 95°C. After cooling down to RT, the specimens were washed in deionized (DI) water for 5 min each and then equilibrated with PBS for 1 h. The primary antibodies were diluted in PBST + 0.01% w/v NaN3 , and sample incubation was performed at 37°C in gentle shaking. After washing with PBST at 37°C for 24 h, the stained samples were incubated in gentle shaking with the secondary antibodies at 37°C in PBST + 0.01% w/v NaN3. Next, the stained samples were extensively washed with PBST at 37°C, equilibrated first in 30% TDE/PBS (v/v), then in 68% TDE/PBS (v/v), and finally placed in the sandwich with 68% TDE.
Application to iDISCO method
Single deparaffinized slices with a thickness of 500 μm were processed with a modified iDISCO protocol. Specimens were dehydrated with increasing concentrations of methanol MeOH/H2O (20%, 40%, 60%, 80%, and 100%), with gentle rotation for 1 h each at RT. Samples were then incubated in 66% dichloromethane (DCM) / 33% MeOH overnight at RT with gentle rotation. Afterwards, samples were washed with 100% MeOH for 1 h twice, and autofluorescence was bleached through incubation with 5% H2O2 in MeOH at 4°C overnight, without shaking. After bleaching, samples were rehydrated with decreasing concentrations of MeOH/H2O (80%, 60%, 40%, 20%), followed by one PBS and two PBST (1× PBS and 0.2% v/v Triton X-100) washes, all with gentle rotation for 1 h each at RT. On the same day, samples were incubated first with the Permeabilization solution (1× PBS, 0.2% v/v Triton X-100, 20% v/v DMSO and 0.3M glycine), followed by incubation in Blocking solution (1× PBS, 0.2% v/v Triton X-100, 0.2% w/v gelatin porcin skin and 0.01% w/v NaN3), both for 24 h, at 37°C with gentle rotation. Primary antibodies were diluted in PBSTW-Heparin (1× PBS, 0.2% v/v TWEEN-20 and 0.01 mg/ml Heparin) for 6 days, at 37°C with gentle rotation. Samples were then washed for 1 day in PBSTw-Heparin and then incubated with the secondary antibodies in PBSTw-Heparin for 4 days, at 37°C with gentle rotation. After 1 day of washes in PBSTw-Heparin, samples were dehydrated as previously described and incubated for 3 h in 66% DCM / 33% MeOH. Finally, they were washed in 100% DCM for 30 min twice and incubated and stored in DBE (Dibenzyl ether) at RT.
Protocol references
Quantitative cytoarchitectural phenotyping of deparaffinized human brain tissues
Danila Di Meo, Michele Sorelli, Josephine Ramazzotti, Franco Cheli, Samuel Bradley, Laura Perego, Beatrice Lorenzon, Giacomo Mazzamuto, Aron Emmi, Andrea Porzionato, Raffaele De Caro, Rita Garbelli, Dalila Biancheri, Cristiana Pelorosso, Valerio Conti, Renzo Guerrini, Francesco S. Pavone, Irene Costantini
bioRxiv 2024.09.10.612232; doi: https://doi.org/10.1101/2024.09.10.612232
Acknowledgements
This project received funding from the European Union’s Horizon 2020 research and innovation Framework Program under grant agreement No. 654148 (Laserlab-Europe), from HORIZON-INFRA-2022-SERV-B-01
“EBRAINS 2.0: A Research Infrastructure to Advance Neuroscience and Brain Health” Horizon Europe – Framework Programme for Research and Innovation (2021-2027). This research has also been supported by the Italian Ministry for University and Research in the framework of the Advanced Light Microscopy Italian Node of Euro-Bioimaging ERIC and by the European Union – Next Generation EU, Mission 4 Component 1, CUP B53C22001810006, Project IR0000023 SeeLife Strengthening the Italian Infrastructure of Euro-Bioimaging. From the General Hospital Corporation Center of the National Institutes of Health under award number U01 MH117023 and BRAIN CONNECTS (award number U01 NS132181). The content of this work is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health-USA. The work was also supported by Fondazione Cassa di Risparmio di Firenze (project Human Brain Optical Mapping).