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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

Abstract

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Introduction

Analyzing the 3D molecular organization and cytoarchitecture of large volumes of the human brain requires optical transparency of specimens, achieved through protocols with extensive processing time. Optical clearing techniques were developed to minimize heterogeneity in refractive index (RI) within the tissues, thereby reducing light scattering and increasing the light penetration depth for high-resolution imaging1,2,3,4,5. Current advances in clearing and deep tissue-labeling methods allow volumetric imaging of intact rodent organs and embryos by exploiting cutting-edge microscopy techniques6,7,8,9,10,11,12.

However, volumetric 3D reconstruction of large areas of the postmortem human brain still represents a challenging task compared with model organisms. The complex biological composition and the variable postmortem fixation and storage conditions can compromise the tissue clearing efficiency, the antibody penetration depth, and the epitope recognition13,14,15,16,17,18,19. Moreover, mechanical tissue sectioning and subsequent clearing and labeling of each slice is still required to achieve an efficient clearing and uniform labeling of large human brain areas, resulting in long processing times and the need for sophisticated custom equipment, compared with model organisms15,20,21,22.

The SWITCH - H2O2 - antigen Retrieval -TDE (SHORT) tissue transformation technique has been developed specifically to analyze large volumes of the human brain18,23. This method employs the tissue structural preservation of the SWITCH protocol11 and high concentrations of peroxide hydrogen to decrease tissue autofluorescence, in combination with epitope restoration. SHORT allows uniform staining of human brain slices with markers for different neuronal subtypes, glial cells, vasculature, and myelinated fibers18,24. Its results are compatible with the analysis of both low- and high-density proteins. The resulting high transparency levels and uniform labeling enable volumetric reconstruction of thick slices with fluorescence microscopy, in particular, for fast acquisition light-sheet apparatus can be used18,24,25,26,27.

In this work, we describe how the SHORT tissue transformation technique can be used for simultaneous clearing and multi-round labeling of tens of formalin-fixed human brain sections. Four different fluorescent markers can be used together, leading to the identification of different cellular sub-populations. After clearing, high-resolution volumetric imaging can be performed with fluorescence microscopy. Here, we used a custom-made inverted LSFM18,24,25,26,27, which enables fast optical sectioning of the sample and rapid acquisition of multiple channels in parallel. With this routinely high-throughput protocol, it is possible to obtain a comprehensive cellular and structural characterization with a sub-cellular resolution of large areas of the human brain as already demonstrated in the mapping of an entire Broca's area23.

Protocol

Formalin-fixed human tissue samples were provided by the Department of Neuropathology at the Massachusetts General Hospital (MGH) Autopsy Service (Boston, USA). Written consent was obtained from healthy participants prior to death, following IRB-approved tissue collection protocols from the Partners Institutional Biosafety Committee (PIBC, protocol 2003P001937). The authorization documents are kept with the MGH Autopsy Services in Boston, MA, United States, and are available upon request.

1. Agarose embedding and sample cutting

  1. Prepare a 4% w/v agarose solution in 1x phosphate-buffered saline (PBS, pH 7.4) in a beaker and embed the tissue block inside. Wait until it reaches room temperature (RT) and then store at 4 °C for 24 h.
  2. To obtain high-precision sections, glue the sample to the vibratome's specimen disc and fill the tray with cold 1x PBS (pH 7.4). Adjust the vibratome parameters such as frequency, amplitude, and speed according to the type of tissue to be sliced (values used: frequency = 5 (50 Hz); amplitude = 0.4; speed = 3 (0.15 mm/s).
    NOTE: Different vibratomes should be used depending on the sample volume. For samples with a maximum volume of 70 x 40 x 15 mm, a vibratome (e.g., Leica VT1000 S) can be used; for larger samples, it is possible to use a compresstome (e.g., VF-900-0Z Microtome18) or a custom-made apparatus21. Here, we present slices cut with a thickness of either 400 µm or 500 µm.
  3. After cutting, put all the slices in a preserver solution composed of 1x PBS (pH 7.4) with 0.01% w/v Sodium Azide (NaN3) and store at 4 °C.
    ​NOTE: Before embedding, wait until the agarose solution reaches 37 °C. A higher temperature might damage the specimen. It is recommended not to leave the samples in PFA for more than 48 h as prolonged fixation might interfere with the labeling procedure by damaging antigens and increasing tissue autofluorescence. For long-term storage of human tissues, PBS with NaN3 is used to prevent microbial contamination. Due to the toxicity of NaN3, prepare the preserver solution under a chemical hood.

2. Tissue fixation

NOTE: All the solutions used in the following protocol are prepared in large volumes, sufficient to process all the slices of the same tissue block and minimize the technical variability and reduce the time for individual steps.

  1. Prepare the Switch-off solution with 50% v/v 1x 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. Place all the samples in dishes with screw lids (70 x 23 mm) and incubate them with the Switch-off solution with gentle shaking at 4 °C for 1 day, protecting them from light with aluminum foil.
  2. Prepare the Switch-on solution with 1x PBS (pH 7.4) and fresh 1% v/v glutaraldehyde. Place all the samples in new dishes and incubate them with the Switch-on solution with gentle shaking at 4 °C for 1 day, protecting them from light with aluminum foil.
    ​NOTE: Due to the high toxicity and light sensitivity of glutaraldehyde, both Switch-off and Switch-on solutions must always be prepared fresh and kept under the chemical hood on ice. Always fill the dish with 20 mL of each solution and seal it with parafilm.

3. Inactivation and clearing

NOTE: Reactive glutaraldehyde in the samples must be inactivated by incubation with an inactivation solution consisting of 1x PBS pH 7.4, 4% w/v acetamide, 4% w/v glycine, pH 9.0. The solution can be stored at 4 °C for up to 3 months. To remove lipids and make the tissue transparent, we use the clearing solution consisting of 200 mM sodium dodecyl sulfate (SDS), 20 mM sodium sulfite (Na2SO3), and 20 mM boric acid (H3BO3), pH 9.0. The solution must be stored at RT as SDS precipitates at 4 °C. All the following steps will be done in tubes filled with the solution.

  1. Move the samples from the dishes to tubes and wash for 3 x 2 h with 1x PBS pH 7.4 at RT in gentle shaking.
    NOTE: For step 3.1, it is necessary to work under the chemical hood because of the glutaraldehyde toxicity. The acetamide, SDS, and boric acid are also toxic reagents, and they must be handled under a chemical hood. Always fill the tube with 50 mL of each solution.
  2. Incubate the samples in inactivation solution at 37 °C in a water bath overnight (o/n).
  3. Wash for 3 x 2 h in 1x PBS pH 7.4 at RT in gentle shaking.
  4. Incubate the samples in clearing solution at 55 °C in a water bath for 3-7 days. Change the solution every 2 days (Table 1).

4. Immunolabeling

NOTE: Before the labeling step, it is necessary to remove the residual SDS, reduce the autofluorescence, and unmask the epitopes with an antigen retrieval solution consisting of 10 mM Tris base, 1 mM EDTA, and 0.05% v/v Tween 20, pH 9. The solution's pH of 9 is optimized for an efficient retrieval process; Tris base acts as a buffering agent to maintain a stable pH environment; EDTA, which is a chelating agent, enhances the antigen's accessibility. The nonionic detergent Tween 20 aids in improving the permeability of the tissue. This solution can be stored at 4 °C. It is important to note that hydrogen peroxide combined with the antigen retrieval step have a synergistic effect in reducing the autofluorescence signal, by breaking down and/or modifying the endogenous fluorophores responsible for non-specific background signal (such as lipofuscin).

  1. Prepare 1x PBS with 0.1% v/v Triton X-100 (PBST), prewarm it to 37 °C, and wash the samples 3 x 3 h during the day in gentle shaking at 37 °C in the incubator. Leave the last wash o/n.
    NOTE: Store the PBST stock solution at 4 °C. It is crucial to remove SDS from the tissue as it can form insoluble precipitates at low temperatures that may interfere with subsequent acquisition processes.
  2. The day after, add 10 mL of 30% v/v hydrogen peroxide (H2O2) to each sample and leave it with gentle shaking at RT for 45 min.
  3. Wash 3 x 10 min with 1x PBS (pH 7.4) with gentle shaking at RT.
  4. Preheat the antigen retrieval solution at 95 °C in a water bath; transfer the samples to the heated solution and leave them at 95 °C for 10 min.
  5. Allow the specimens to cool down to RT for 40 min in gentle shaking.
  6. Wash 3 x 5 min with deionized water in gentle shaking.
  7. Equilibrate the samples in PBS at 4 °C for 15 min.
  8. Prepare fresh antibody solution made of PBST with 0.01% w/v NaN3 (see the NOTE for step 1.3) and add primary antibodies. Incubate the samples with the solution in dishes with screw lids at 37 °C for n days (1-7) in an incubator with gentle shaking and protection from light (Table 2).
    NOTE: The incubation time is size- and thick-dependent (Table 1 and Table 2).
  9. After n days, move the samples into tubes and wash 3 x 2 h with prewarmed PBST at 37 °C with gentle shaking in the incubator.
  10. Add secondary antibodies in PBST with 0.01% w/v NaN3 and incubate the samples in new dishes with screw lids at 37 °C for n days (1-6) in an incubator with gentle shaking and protected from light (Table 1 and Table 2).
  11. After n days, transfer the samples to tubes and wash 3 x 3 h during the day with prewarmed PBST at 37 °C in a gentle shaking incubator. Leave the last wash o/n.
    ​NOTE: As Triton X-100 and Tween 20 are viscous, pipette slowly to allow the tip to fill. Always fill the tube with 50 mL of each solution. Use 10 mL of antibody solutions for each sample and seal the dish with parafilm to prevent evaporation of the solution. Since the antibody solutions contain NaN3, they can be stored at 4 °C and reused to label new specimens within 2 weeks.

5. Refractive index matching

NOTE: To achieve a high level of transparency, it is necessary to homogenize the tissue refractive index. Here we use 2,2'-thiodiethanol (TDE) diluted in 1x PBS. TDE solutions must be stored at RT.

  1. Transfer the samples to new dishes, add 30% v/v TDE, and leave them for 4 h with gentle shaking at RT.
  2. Remove the 30% v/v TDE solution, add 68% v/v TDE to the samples, and leave them with gentle shaking at RT.
    ​NOTE: As TDE is viscous, pipette slowly to allow the tip to fill. Inhalation of TDE vapor or mist can irritate the respiratory system. Therefore, it is advisable to work in a well-ventilated area or use fume hoods to minimize exposure. TDE solutions must be stored at RT. Use 10 mL of TDE solutions for each sample.

6. Sample mounting

NOTE: To facilitate sample mounting and LSFM image acquisition, we use a custom-made, sealed sample holder (termed "sandwich"), which consists of three parts: a microscope slide, a spacer, and a coverslip.

  1. Put the steel spacer (56 x 56 x 0.5 mm3) over the microscope slide (60 x 60 x 1 mm3) and lay the sample on the microscopy slide.
  2. Carefully put the quartz coverslip (60 x 60 x 0.5 mm3), clip everything together, and prepare a two-component silicon glue in a 1:1 ratio. Use a quartz coverslip to match the refractive index and reduce the optical aberration during the acquisition process.
  3. Using a 1 mL syringe, fill the space between the spacer and the coverslip with a two-component silicon glue and let it dry for 10 min.
  4. Remove the clips and use a small needle to slowly fill the entire sandwich with 68% v/v TDE.
  5. Make fresh glue and seal the sandwich.
    ​NOTE: If the TDE accidentally reaches the spacer in step 6.1, the glue will not cure. Make sure that no air bubbles form during step 6.2.

7. Stripping

NOTE: The structural preservation and the enhanced epitope accessibility of SHORT allow multi-round labeling of slices by removing the antibodies and restaining the samples with other markers.

  1. Open the sandwich with a blade, put the specimens in new dishes with screw lids filled with 30% v/v TDE, and leave them for 3 h.
  2. Transfer the samples to tubes, wash for 3 x 2 h in 1x PBS (pH 7.4) at RT with gentle shaking, and leave the last wash o/n.
  3. Place the samples in clearing solution in a water bath at 80 °C for 4 h.
  4. Wash for 3 x 2 h in 1x PBST (pH 7.4) at 37 °C in gentle shaking and leave the last wash o/n. Now the sample is ready to be labeled again starting from step 4.8.
    NOTE: Always fill the tube with 50 mL of each solution. Use 10 mL of TDE solution for each sample.

Results

The protocol described here enables the simultaneous treatment of multiple slices, ranging in thickness from 100 µm to 500 µm, using the SHORT method. This approach significantly reduces the overall processing time for the entire procedure. In this work, we provide a comprehensive description of the entire pipeline (Figure 1) for processing multiple postmortem human brain thick sections simultaneously and we demonstrate the protocol on 24 slices at once (Figure...

Discussion

High-resolution imaging and 3D reconstruction of large human brain areas require mechanical tissue sectioning followed by optical clearing and immunolabeling of single slices. The protocol presented here describes how the SHORT tissue transformation method can be used for rapid and simultaneous processing of multiple human brain thick sections for 3D brain reconstruction with a subcellular resolution with LSFM.

Unlike other approaches, with the SHORT method the clearing and multi-labeling step...

Disclosures

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgements

We thank Bruce Fischl, Massachusetts General Hospital, A.A. Martinos Center for Biomedical Imaging, Department of Radiology, for providing the human brain specimens analyzed in this study. This project received funding from the European Union's Horizon 2020 Research and Innovation Framework Programme under grant agreement No. 654148 (Laserlab-Europe), from the European Union's Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2) and No. 945539 (Human Brain Project SGA3), from the General Hospital Corporation Center of the National Institutes of Health under award number U01 MH117023, and from the Italian Ministry for Education in the framework of Euro-Bioimaging Italian Node (ESFRI research infrastructure). Finally, this research was carried out with the contribution of "Fondazione CR Firenze." 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. Figure 1 was created with BioRender.com.

Materials

NameCompanyCatalog NumberComments
2,2'-thiodiethanolMerck Life Science S.R.L.166782
Acetamide >= 99.0% (GC)Merck Life Science S.R.L.160
Agarose High EEOMerck Life Science S.R.L.A9793
Boric AcidMerck Life Science S.R.L.B7901
Compressome VF-900-0Z MicrotomePrecisionary/
CoverslipsLaserOptex/customized
Ethylenediaminetetraacetic acid disodium salt dihydrateMerck Life Science S.R.L.E5134
GlutaraldehydeMerck Life Science S.R.L.G7651
GlycineSanta Cruz BiotechnologySC_29096
Hydrogen Peroxide 30%Merck Life Science S.R.L.
Incubator ISS-4075Lab companion /
Light-sheet fluorescence microscopy (LSFM)//custom-made
Loctite AttakHenkel Italia srl/
Microscope slidesLaborchimica/customized
Phospate buffer saline tabletMerck Life Science S.R.L.P4417
Picodent TwinsilPicodent13005002out of production
Potassium Hydrogen PhtalateMerck Life Science S.R.L.P1088
Sodium AzideMerck Life Science S.R.L.S2002
Sodium Dodecyl SulfateMerck Life Science S.R.L.L3771
Sodium SulfiteMerck Life Science S.R.L.S0505
SpacersMicrolaser srlcustomized
Sputum Containers (dishes with screw lids)Paul Boettger GmbH & Co. KG07.061.2000
Tris BasePanReac AppliChem (ITW reagents)A4577,0500
Triton X-100Merck Life Science S.R.L.T8787
TubesSarstedt62 547254
Tween 20Merck Life Science S.R.L.P9416
Vibratome VT1000SLeica Biosystem/
Water bath MemmertWNB 7-45
Antibodies and Dyes
Alexa Fluor 488 AffiniPure Alpaca Anti-Rabbit IgG (H+L)Jackson Immuno Reasearch611-545-215Dilution used, 1:200
Alexa Fluor 488 AffiniPure Bovine Anti-Goat IgG (H+L)Jackson Immuno Reasearch805-545-180Dilution used, 1:200
Alexa Fluor 647 AffiniPure Alpaca Anti-Rabbit IgG (H+L)Jackson Immuno Reasearch611-605-215Dilution used, 1:200
Anti-NeuN AntibodyMerck Life Science S.R.L.ABN91Dilution used, 1:100
Anti-Parvalbumin antibody (PV)Abcamab32895Dilution used, 1:200
Anti-Vimentin antibody [V9] - Cytoskeleton Marker (VIM)Abcamab8069Dilution used, 1:200
Calretinin Polyclonal antibodyProteinTech12278_1_APDilution used, 1:200
DAPIThermoFisherD3571Dilution used, 1:100
Donkey Anti-Mouse IgG H&L (Alexa Fluor 568)Abcamab175700Dilution used, 1:200
Donkey Anti-Mouse IgG H&L (Alexa Fluor 647)Abcamab150107Dilution used, 1:200
Donkey Anti-Rabbit IgG H&L (Alexa Fluor 568)Abcamab175470Dilution used, 1:200
Donkey Anti-Rat IgG H&L (Alexa Fluor 568) preadsorbedAbcamab175475Dilution used, 1:200
Goat Anti-Chicken IgY H&L (Alexa Fluor 488)Abcamab150169Dilution used, 1:500
Goat Anti-Chicken IgY H&L (Alexa Fluor 568)Abcamab175711Dilution used, 1:500
Goat Anti-Chicken IgY H&L (Alexa Fluor 647)Abcamab150171Dilution used, 1:500
Goat Anti-Rabbit IgG H&L (Alexa Fluor 488)Abcamab150077Dilution used, 1:200
Recombinant Alexa Fluor 488 Anti-GFAP antibodyAbcamab194324Dilution used, 1:200
Somatostatin Antibody YC7Santa Cruz Biotechnologysc-47706Dilution used, 1:200
Vasoactive intestinal peptide (VIP)ProteinTech16233-1-APDilution used, 1:200

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Optical ClearingLight sheet Fluorescence MicroscopyHuman Brain Imaging3D ReconstructionCytoarchitectureTissue TransformationSHORT ProtocolNeuronal MarkersMulti labelingAutofluorescent SignalsHigh throughput ProtocolPostmortem ProcessingSubcellular Resolution

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