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. Author manuscript; available in PMC: 2024 Jul 12.
Published in final edited form as: Methods Mol Biol. 2023;2561:87–101. doi: 10.1007/978-1-0716-2655-9_5

Three-dimensional Imaging of Fibrinogen and Neurovascular Alterations in Alzheimer’s Disease

Mario Merlini 1,2,*, Elif G Sozmen 1,2,3, Keshav S Subramanian 2, Alissa L Nana 3, William W Seeley 3, Katerina Akassoglou 1,2,3
PMCID: PMC11243589  NIHMSID: NIHMS2007575  PMID: 36399266

Abstract

Cerebrovascular dysfunction is a hallmark of Alzheimer’s disease (AD) that is linked to cognitive decline. However, blood-brain barrier (BBB) disruption in AD is focal and requires sensitive methods to detect extravasated blood proteins and vasculature in large brain volumes. Fibrinogen, a blood coagulation factor, is deposited in AD brains at sites of BBB disruption and cerebrovascular damage. This chapter presents the methodology of fibrinogen immunolabeling-enabled three-dimensional (3-D) imaging of solvent-cleared organs (iDISCO) which, when combined with immunolabeling of amyloid β (Aβ) and vasculature, enables sensitive detection of focal BBB vascular abnormalities, and reveals the spatial distribution of Aβ plaques and fibrin deposits, in large tissue volumes from cleared human brains. Overall, fibrinogen iDISCO enables the investigation of neurovascular and neuroimmune mechanisms driving neurodegeneration in disease.

Keywords: iDISCO, Fibrinogen, Blood-brain Barrier, Biomarker, Alzheimer’s disease (AD), Tissue Clearing, Microscopy, Neurodegeneration, Neurovascular, Neuroinflammation

1. Introduction

Cerebrovascular damage, microbleeds, blood-brain barrier (BBB) disruption and fibrinogen deposition are features of Alzheimer’s disease (AD) pathology [1–6]. Given the contribution of cerebrovascular dysfunction to cognitive decline in AD [4–8], sensitive methods to detect cerebrovascular damage in large tissue volumes are critical for the understanding of the onset and progression of neurodegeneration, as well as the effects of therapies on the BBB. Recent advances in tissue-clearing methods, combined with immunolabeling, have enabled three-dimensional (3-D) imaging of vasculature by fluorescence microscopy using iDISCO [9–11]. However, iDISCO imaging of brain vasculature alone does not capture plasma protein extravasation into the CNS. Here we describe fibrinogen iDISCO as a highly sensitive imaging method for BBB disruption in human brains.

Fibrinogen is deposited in the AD brain at sites of neurovascular abnormalities and neuroinflammation [7, 12, 13]. It increases in the brain and the cerebrospinal fluid (CSF) of AD patients who are carriers for ApoE4, the major genetic risk factor for AD [5, 14]. Fibrinogen activates microglia and is deposited at sites of microglial activation in human AD brains [7, 8, 12, 13, 15]; this activation promotes oxidative stress and spine elimination, which cause cognitive impairment in mouse models of AD [7, 16]. Fibrinogen is unique among other blood proteins in that it clots and forms insoluble fibrin deposits in the CNS. It binds amyloid β (Aβ), leading to inhibition of fibrin degradation and, therefore, sustained fibrin deposits [17].

For fibrinogen iDISCO, the areas of interest in the human brain must be dissected and fixed in formalin. The tissue is then sectioned, dehydrated, and immunolabeled for fibrinogen, Aβ, and a marker of the vasculature, then cleared and imaged. Fibrinogen iDISCO reveals distinct patterns of BBB abnormalities in AD brains, including focal fibrin deposits around tortuous vascular structures and deposits in the brain parenchyma, together with or independent of Aβ deposition [3, 7]. Thus, fibrinogen iDISCO may be used for the detection of BBB abnormalities during aging, or in AD and other neurological diseases, including multiple sclerosis, traumatic brain injury, stroke, epilepsy and psychiatric disorders.

We describe a protocol for triple-immunolabeling of fibrinogen, Aβ, and vasculature in human brain tissue, using iDISCO. The iDISCO protocol for human brains described herein was developed based on the iDISCO protocol for mouse tissue [10] described in: http://idisco.info.

2. Materials

2.1. Human Tissue Fixation and Processing

  1. Neutral-buffered formalin, 10%, 50 mL per tissue block.

  2. 10X PBS stock, approximately 400 mL per assay. 1X working concentration contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4. Sodium azide, 0.02% in 1X PBS prepared with double-distilled water (ddH2O), 200 mL per tissue block.

  3. Forceps, flat-tipped, non-serrated.

  4. Specimen jars, 50–100 mL, flat-bottomed.

2.2. Tissue Sectioning and Dehydration

  1. Eppendorf tubes, 5 mL.

  2. Surgical scalpel, #11, disposable.

  3. Nutator mixer.

  4. Methanol (MeOH), 100% (20 mL per sample).

  5. MeOH dilution series in ddH2O, 5 mL each: 20%, 40%, 60%, and 80%, kept at room temperature (RT).

  6. Syringes, disposable, 10 to 20 cc.

  7. Disposable needles, 18–20 gauge.

  8. Dichloromethane (DCM)/methanol (MeOH) solution, 66%/33%, freshly prepared (5 mL per sample), kept at RT.

  9. Hydrogen peroxide (H2O2), 5% in methanol, freshly prepared (5 mL per sample) and kept at 4°C.

2.3. Immunolabeling

  1. Incubator, 37°C, in which the nutator mixer can be placed.

  2. Eppendorf tubes, 5 mL.

  3. MeOH dilution series in ddH2O, 5 mL each: 20%, 40%, 60%, and 80%, kept at room temperature (RT).

  4. 10X PBS..1X working concentration contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4.

  5. Triton X-100.

  6. Tween-20.

  7. Glycine.

  8. Dimethylsulfoxide (DMSO)

  9. Heparin, 10 mg/mL in ddH2O. Can be stored for 6 months at 4°C, prepare 10 mL of stock solution.

  10. Normal donkey serum.

  11. PTx.2 washing solution (1 L): 10X PBS, 100 mL, Triton X-100, 2 mL, to 1 L with ddH2O. Can be stored for 6 mo at RT.

  12. Permeabilization Solution (500 mL): PTx.2, 400 mL, glycine, 11.5 g, DMSO, 100 mL, prepared fresh for each experiment and kept at RT.

  13. Blocking solution (50 mL for 5 mL per sample): PTx.2, 42 mL, normal donkey serum, 3 mL, DMSO, 5 mL. Can be prepared 7 d ahead of each experiment, kept at 4°C.

  14. PTwH: 10X PBS, 100 mL, Tween-20, 2 mL, heparin, 1 mL of a 10 mg/mL solution, to 1 L with ddH2O. Can be stored for 6 mo at RT.

  15. Primary antibody solution (5 mL per sample): DMSO, 5%, donkey serum, 3% in PTwH. Can be prepared 7 d ahead of each experiment and kept at 4°C.

  16. Secondary antibody solution (5 mL per sample): donkey serum, 3% in PTwH. Can be prepared 7 d ahead of each experiment and kept at 4°C.

  17. Guinea pig anti-human CD31/PECAM-1 antibody (Synaptic Systems), diluted 1:100 to add 20 µL of antibody per sample. The antibody must be from the indicated supplier (see Note 5).

  18. Rabbit anti-human β amyloid antibody (IBL-America), 20 µL per sample (1 µg/ml final concentration). The antibody must be from the indicated supplier (see Note 5).

  19. Sheep anti-human fibrinogen (US Biological), 20 µL per sample (30 µg/ml final concentration). The antibody must be from the indicated supplier (see Note 5).

  20. Alexa 488-conjugated, donkey anti-guinea pig secondary antibody (Jackson ImmunoResearch) 40 µL per sample (10 µg/mL final concentration) The antibody must be from the indicated supplier(see Note 6).

  21. Alexa 647-conjugated, donkey anti-rabbit secondary antibody (Jackson ImmunoResearch) 40 µL per sample (10 µg/mL final concentration). The antibody must be from the indicated supplier (see Note 6).

  22. Cy3-conjugated, donkey anti-sheep secondary antibody (Jackson ImmunoResearch) 40 µL per sample (10 µg/mL final concentration). The antibody must be from the indicated supplier (see Note 6).

2.4. Tissue Clearing

  1. Methanol (MeOH), 100% (20 mL).

  2. MeOH dilution series in ddH2O, 5 mL each: 20%, 40%, 60%, and 80%, kept at RT.

  3. Disposable syringe, 10 to 20 cc.

  4. Disposable needles, 18–20 gauge.

  5. Dichloromethane (DCM)/methanol (MeOH) solution, 66%/33%, freshly prepared (5 mL per sample), kept at RT.

  6. DCM, 100%, 10 mL per sample.

  7. Dibenzyl ether (DBE), 5 mL per sample.

2.5. Image Acquisition and Processing

  1. Caps, flat-topped, for 15-mL centrifuge tubes.

  2. Kwik-Sil® silicone adhesive, or equivalent.

  3. Coverslip, glass, square or circular.

  4. Kimwipes®.

  5. Pipets, disposable, 2 mL.

  6. Olympus FluoView 1000 MPE platform, or equivalent (see Note 11).

  7. Image J software.

3. Methods

8–10 mm thick coronal slabs of brains from AD patients and controls were obtained as previously described [18] (see Note 1)

3.1. Fixation of Human Brain Tissue

  1. Immerse coronal slabs in 10% buffered formalin for 72 h at RT. Ensure slabs are placed flat and arranged in anatomical position during fixation, using flat-tipped forceps to minimize mechanical damage.

  2. After 72 h, rinse the slabs 3 times in 0.02% sodium azide, then store them in 0.02% sodium azide at 4°C.

  3. Use a scalpel to cut 5–10-mm-thick blocks (approximately 1×2 cm) of the region of interest from the fixed coronal slabs, including dementia-related regions [20].

Place tissue blocks into 50–100 mL flat-bottomed specimen jars and store in 0.02% sodium azide at 4°C until needed.

3.2. Tissue Sectioning and Dehydration

  1. Perform all steps described below in 5-mL Eppendorf tubes. Cut the formalin-fixed tissue blocks into 1.5–2-mm-thick sections with a scalpel (see Note 2).

  2. Dehydrate the tissue in the following freshly prepared, consecutive MeOH/ddH2O solutions, for 1 h each at RT: 20%, 40%, 60%, 80%, 100%.

  3. Cool the tissues at 4°C for 1 h (see Note 3).

  4. Incubate the tissues in DCM/MeOH 66/33% on a nutator mixer at RT, overnight. Use large-bore needle attached to the disposable syringe to retrieve DCM from the secure cap container.

  5. Wash the samples twice in 100% MeOH for 30 min at RT on the nutator mixer, then cool the samples at 4°C for 1 h.

  6. Bleach the brain tissue samples in freshly made, pre-cooled (4°C) 5% H2O2 in MeOH for 36 h at 4°C.

3.3. Immunolabeling

Use 2-mL Eppendorf tubes for the primary and secondary antibody incubations (Steps 3 and 5, below); use 5-mL Eppendorf tubes for all other steps to ensure sufficient coverage. For larger tissue samples, use 5 mL tubes (see Notes 4–6).

  1. Rehydrate the samples in the following consecutive MeOH solutions for 1 h each: 80%, 60%, 40%, 20%, ddH2O.

  2. Wash samples in PTx.2 twice for 1 h at RT under continuous shaking.

  3. Incubate the samples in Permeabilization solution at 37°C for 36 h on the nutator mixer.

  4. Incubate the samples in Blocking solution at 37°C for 48 h on nutator mixer.

  5. Incubate the samples in Primary antibody solution containing sheep anti-human fibrinogen, rabbit anti-human β amyloid, guinea pig anti-human CD31/PECAM-1 (all antibodies at a 1:100 dilution), at 37°C for 72 h on the nutator mixer (see Note 5).

  6. Wash the samples in PTwH 5 times on the nutator mixer, changing the solution every 2 h. Leave in PTwH overnight at 37°C on the nutator mixer.

  7. Incubate the samples in Secondary antibody solution containing Alexa 488-conjugated donkey anti-guinea pig, Alexa 647-conjugated donkey anti-rabbit, and Cy3-conjugated donkey anti-sheep antibodies (all at 1:200 dilution) at 37°C for 72 h on the nutator mixer (see Note 6).

  8. Wash the samples in PTwH 5 times at RT on the nutator mixer, changing the solution every 2 h. Leave in PTwH overnight at RT on the nutator mixer.

3.4. Tissue Clearing

Perform all steps described below in 5-mL Eppendorf tubes (see Note 4).

  1. Dehydrate the tissue in the following freshly prepared, consecutive MeOH/ddH2O solutions, for 1 h each: 20%, 40%, 60%, 80%, 100%, at RT (see Note 7).

  2. Using forceps, transfer the samples into freshly prepared DCM/MeOH 66/33%. Incubate on the nutator mixer at RT for 3 h.

  3. Wash in 100% DCM twice for 30 min each, on the nutator mixer at RT. Decant DCM between washes.

  4. Using forceps, carefully place the samples in 100% DBE-containing (new) Eppendorf tubes. Gently invert the tubes to ensure adequate mixing of the DBE. The samples will become translucent within 30–60 s (see Note 8).

  5. Store the cleared tissue at RT in an upright position and shield from light. Ensure that the Eppendorf tubes are fully filled and closed to prevent oxidation of the samples. Samples can be stored for several months without an appreciable decrease in quality.

3.5. Preparation of Imaging Chambers for Image Acquisition

The tissue samples must be immersed in DBE at all times, including during imaging. Therefore, an imaging chamber containing DBE and the sample must be prepared before acquiring images.

  1. Flat-topped screw caps of 15-mL centrifuge tubes will serve as imaging chambers for the tissue samples. Prepare separate imaging chambers for each sample. Alternatively, the lids of 5-mL Eppendorf tubes can be used for smaller samples.

  2. Fill each lid with 100% DBE using a disposable pipet.

  3. With a pair of forceps, gently remove the sample from its storage tube and place in the DBE-filled lid prepared in Step 2.

  4. Add extra DBE to the lid until the tissue is completely submerged.

  5. Place a square or circular glass coverslip on top of the filled lid. The size of the coverslip should be larger than the lid. Make sure no air bubbles become trapped between the coverslip and the samples (see Note 9).

  6. Seal the coverslip onto the lid using Kwik-Sil® silicone adhesive, verifying that there are no DBE leaks from the seal. Carefully clean the coverslip glass and the lid, removing all traces of DBE with a Kim-wipe (see Notes 10 and 11).

3.6. Image Acquisition and Processing

  1. Use the 543-nm HeNe gas laser to excite Cy3-labeled secondary antibodies and the 635-nm diode laser to excite Alexa 647-labeled secondary antibodies. Tune the two-photon laser to 940 nm to excite Alexa 488-labeled secondary antibodies (see Notes 12 and 13).

  2. Separate the Cy3 (543 nm) and Alexa 647 (635 nm) fluorescence emission spectra using a 640-nm dichroic mirror, with a 560–620-nm (Cy3) and a 655–755-nm (Alexa 647) emission filter mounted before the detector. For the Alexa 488 secondary antibodies, pass the fluorescence emission from the two-photon laser through a 495–540-nm emission filter mounted before the non-descanned detector.

  3. Acquire Z-stacks in the sequential imaging mode at a 1-µm step resolution, 4.0-µs pixel dwell-time, and 512 × 512 or 1024 × 1024 pixel resolution.

  4. Process images with ImageJ software. Load the combined Alexa 488, Cy3, and Alexa 647 image Z-stack files as separate Z-stacks into ImageJ. Convert images to 8-bit grey-scale, and de-noise the images using the “Remove outliers” plugin with the radius set to 2.0 pixels and the threshold set to 50 internal units. Subtract the Alexa 488 image stack (CD31 signal) from the Cy3 image stack (fibrinogen signal) to ensure removal of possible non-specific, false-positive fluorescence signals arising from lipofuscin and other non-fibrinogen deposits.

  5. Combine the processed-image Z-stacks as pseudo-colored RGB stacks, then re-process using the “Unsharp mask” plugin with the radius/sigma set between 3.0 and 5.0 and the mask weight set at 1/10th of the radius/sigma, e.g., the radius/sigma = 4.0 and the mask weight = 0.4.

  6. Process the RGB stacks into 3-D volume projections using the “3D Viewer” plugin followed by a 360° rotation recording, to yield a .tiff 3-D volume projection image file (see Note 15).

3.7. Qualitative Analysis of iDISCO-processed Tissue

  1. Following image acquisition and processing, analyze the images qualitatively for the presence of fibrinogen in intravascular, vessel-associated, and parenchymal regions, with and without Aβ deposits, as well as tortuous vascular structures [22] with vessel-associated fibrinogen deposits in AD brains using the representative images shown here serving as a guide (Figs. 1A–D and Videos 1–3), (see Notes 1, 16, 17, 18).

  2. Define vessel-associated fibrinogen as vascular or perivascular fibrin deposits present at the abluminal side of CD31-stained vessels in NDCTRL brains using the representative images shown here serving as a guide (Fig. 2 and Video 1), (see Notes 1, 16, 17, 18).

Fig 1. Fibrinogen iDISCO in AD Brain.

Fig 1

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A–D: 3-D immunolabeling of cleared temporo-occipital brain tissue from three AD brains, stained for the vascular marker, CD31 (green), fibrinogen (red) and Aβ (blue). Arrows and asterisks indicate vessel-associated and parenchymal fibrinogen, respectively; # sign indicates intravascular fibrinogen. Magenta in (B) indicates Aβ-associated fibrinogen. E and F: Representative 3-D volume projections of iDISCO-cleared temporo-occipital AD brain tissue, stained for CD31, fibrinogen, and Aβ, showing examples of vascular tortuosity observed in AD brain. Vascular tortuosity was observed in 3 out of the 5 AD brains. Scale bars = 75 µm (A), 25 µm (B), 50 µm (C), and 25 µm (D–F). Images reprinted from Merlini et al [7].

Video 1. Fibrinogen iDISCO in Non-demented Control and AD Brains.

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3-D projection stacks acquired using confocal microscopy, showing fibrinogen (red), CD31-positive blood vessels (green), and Aβ plaques (6E10 antibody, blue). Right side: iDISCO-processed lateral temporo-occipital cortex of an AD patient, showing an example of vascular tortuosity and vessel-associated and parenchymal fibrinogen deposits, as well as Aβ plaques. Left side: iDISCO-processed lateral temporo-occipital cortex of an age-matched NDCTRL subject is shown for comparison. Individual videos reproduced with permission from [7].

Video 3. 3-D imaging of Vascular Tortuosity with Vessel-associated Fibrinogen Deposits in AD Brain.

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iDISCO-processed lateral temporo-occipital cortex showing an example of vascular tortuosity with vessel-associated fibrinogen in an AD brain. 3-D projection (left) and associated Z-stack (right) were acquired using confocal microscopy, and reveal fibrinogen (red), CD31-positive blood vessels (green), and Aβ plaques (6E10 antibody, blue). Individual videos reproduced with permission from [7].

Fig 2. Fibrinogen iDISCO in Non-demented Control Brain Tissue.

Fig 2

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A–D: Representative 3-D volume projections of iDISCO-cleared temporo-occipital brain tissue from age-matched NDCTRL subjects stained for the vascular marker, CD31, fibrinogen, and Aβ. Images are representative from 3 NDCTRL brains. Vessel-associated fibrinogen deposits are present on the abluminal side of CD31-stained vessels. Arrows and asterisks indicate vessel-associated and parenchymal fibrinogen deposits, respectively; # sign indicates intravascular fibrinogen. Scale bars = 50 µm (A and B) and 25 µm (C and D). Images reprinted from Merlini et al. [7].

Video 2. Distinct Patterns of Fibrinogen Deposits in AD Brains.

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iDISCO-processed lateral temporo-occipital cortices of AD patients showing vessel-associated and parenchymal fibrinogen deposit in areas with (left, right) or without (middle) Aβ plaques. 3-D projection stacks acquired using confocal microscopy reveal fibrinogen (red), CD31-positive blood vessels (green), and Aβ plaques (6E10 antibody, blue). Individual videos reproduced with permission from [7].

Acknowledgements

We thank Pamela E. Rios Coronado for expert technical assistance and Kathryn Claiborn for expert editorial assistance. Human tissue samples were provided by the Neurodegenerative Disease Brain Bank at the University of California, San Francisco, which receives funding support from NIH grants P01AG019724 and P50AG023501; the Consortium for Frontotemporal Dementia Research; and the Tau Consortium. The work described herein was supported by a Swiss National Science Foundation Early Postdoc Mobility Fellowship to M.M., the National MS Society Award FAN-2008-37045 to E.G.S. and grants from the Simon Family Trust, the Dagmar Dolby Family Fund, Edward and Pearl Fein, the Conrad N. Hilton Foundation 17348, NIA RF1 AG064926 and NIH/NINDS R35 NS097976 to K.A.

Footnotes

1.

The current protocol has been used for iDISCO processing of blocks of the lateral temporo-occipital cortex dissected from 5 patients with AD and 3 non-demented control (NDCTRL) subjects. The brains were procured and processed according to the UCSF Neurodegenerative Disease Brain Bank protocol [18]. The age range was 70–94 years, female/male percentage was 38%, Braak stage and Thal Phase NDCTRLs: II and 0–2, respectively, for NDCTRLs and IV–VI and 4–5, respectively, for AD. The brains were obtained within a postmortem interval of 6–15 h. Neuropathological diagnoses were made following consensus histological and diagnostic criteria for AD [19–21].

2.

Because of the lack of a species barrier, handle all human tissue using the appropriate personal protective equipment and by performing all steps in a biosafety hood, as required by your institutional guidelines.

3.

Cooling down the brain tissue samples is required to counterbalance the heat generation by the DCM/MeOH.

4.

Perform all steps with tubes fully filled and tightly closed to prevent oxidation of the samples.

5.
If using primary antibodies different from those described in this protocol, studies will be needed to test for compatibility with MeOH and optimal antibody concentration. Although a so-called “alternative method” based on MeOH-free solutions is available (https://idisco.info), in our hands, iDISCO processing of human brain tissue requires the MeOH-based solutions to yield high signal-to-noise ratios during imaging. Use the following method to confirm MeOH compatibility of primary antibodies:
  • Prepare 20-µm-thick frozen sections of the formalin-fixed brain tissue and mount on SuperFrost® slides.
  • Incubate the tissue slides in 100% MeOH for 3 h. Rehydrate the brain tissue directly in PBS and proceed with the regular immunostaining protocol used for staining frozen sections. Use non-MeOH-treated slides as positive controls.
6.

If using secondary antibodies different from those described in this protocol, optimum antibody concentration needs to be determined by the researcher. Always include secondary antibody control tissue samples for each brain in the staining series, i.e., brain tissue samples that are processed along with all other samples from the same brain but for which the primary antibodies are omitted. These controls are essential for evaluating successful staining, determining staining quality in relation to background noise/autofluorescence, and detecting potential false-positive immunofluorescence signals.

7.

As an optional pause in the protocol, the tissues may be left, at this point, in 100% MeOH at RT overnight.

8.

DBE is highly corrosive; take all safety precautionary measures, including adequate personal protective equipment, when handling.

9.

If air bubbles become trapped inside the imaging chamber, they may be removed by slightly lifting the coverslip at a shallow angle. Subsequently, gently add DBE through a 23–27-gauge needle attached to a syringe, until a convex meniscus has formed and the air bubbles are expelled. Gently lower the coverslip and absorb any spilled DBE with a tissue. Seal with Kwik-Sil® as described.

10.

Careful but thorough cleaning of the assembled imaging chamber is required to prevent damage by DBE, to the confocal and two-photon microscope lenses. Use 70% isopropyl alcohol and soft tissues, e.g., KimWipes®.

11.

Do not use any type of glue or silicon sealants, as they will chemically react with DBE, resulting in loss of the seal and subsequent leakage of DBE from the imaging chamber.

12.

We have used the Olympus FluoView 1000 MPE platform, consisting of an Olympus BX61WI microscope with integrated one-photon and two-photon laser lines and an Olympus 20X, 0.5-NA water-immersion lens for fluorescence image acquisition. Any similar type of confocal with a two-photon microscope platform combined with similar lasers and appropriate multi-alkali and/or GaAsP photomultiplier tubes may be used.

13.

One-photon lasers: a 543-nm HeNe gas laser and a 635-nm diode laser. Two-photon laser: Spectra-Physics MaiTai DeepSee eHP, 690–1040 nm Ti:Sapphire femtosecond laser. Dichroic mirror and emission filters: 640-nm dichroic mirror and 495–540-nm, 560–620-nm, and 655–755-nm emission filters. Due to the relatively high photon scattering and autofluorescence in the human brain, especially in white matter, it is highly recommended to excite Alexa 488-labeled proteins/structures of interest with a two-photon laser as described. The Cy3- and Alexa 647-labeled proteins/structures can be imaged with conventional one-photon lasers, as their excitation and emission wavelengths do not significantly overlap with those of brain tissue and, thus, yield less autofluorescence.

14.

Although this protocol describes the use of a combined confocal and two-photon imaging platform for image acquisition, images may be acquired readily with an adequate light-sheet microscope setup that is suitable for volume imaging of DBE-immersed brain tissue.

15.

This Note is also related to Notes 5 and 6. Aged (human) brain has increased intracellular lipofuscin. The pervasive autofluorescence of lipofuscin may also be detected at the longer wavelengths and with the corresponding emission filters described here, and may thus be apparent in the stained brain tissue samples. It is therefore necessary to compare the immunofluorescence signals in each stained sample to those in the corresponding secondary-antibody controls, for proper validation of true- and false-positive signals. The Subtract Background and/or Remove Outliers plugins of ImageJ should be used and set according to the immunofluorescence signals in the respective secondary antibody control samples, for each specific brain.

16.

Neuropathological studies have identified fibrinogen deposition in AD patients, with increased deposition correlating with the ApoE4 mutation [13, 14, 23] and with increased BBB permeability [5]. Fibrinogen iDISCO is ideally suited to decipher the patterns of fibrinogen deposition and cerebrovascular abnormalities in different AD patient populations, and to determine their correlation with the ApoE4 mutation, cerebral amyloid angiopathy, white matter hyperintensities, and microbleeds. As loss of BBB integrity is focal in AD, 3-D imaging of plasma protein extravasation in relatively thick tissue sections is advantageous, compared with methods that rely only on thin tissue sections, since the decreased number, length, and complexity/branching of vascular structures inherent to the latter reduce BBB leak detection, possibly leading to false-negative assessments of BBB permeability. As BBB disruption also occurs focally in other neurological diseases, fibrinogen iDISCO could be applied to neurovascular studies of cortical and white-matter lesions from patients with multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, and some psychiatric disorders [3].

17.

Fibrinogen in the CSF is a biomarker in AD that positively correlates with p-tau and soluble PDGFRβ, a pericyte marker indicative of loss of BBB integrity [5, 24]. Thus, fibrinogen iDISCO in AD brains can be used to co-register neuropathological findings with fluid biomarkers in patients with AD. Furthermore, fibrinogen iDISCO could guide the development of fibrin–PET probes for the direct detection of vascular damage and BBB disruption in human AD brains.

18.

Fibrinogen binds Aβ, and this interaction inhibits fibrin degradation, leading to sustained fibrin deposits [17]. Fibrinogen iDISCO reveals different patterns of fibrin deposits, either perivascular, together with Aβ, or distant from Aβ deposits [7]. Fibrinogen induces microglial activation and correlates with activated microglia in human AD brains [7, 8, 12, 13, 15]. Furthermore, fibrinogen activation of microglia induces the elimination of dendritic spines and causes cognitive impairment in mouse models of AD [7]. Therefore, it could be useful to adapt the fibrinogen iDISCO protocol for triple immunolabeling of fibrinogen, microglia, and vasculature in brains from human AD patients. Overall, 3-D imaging of fibrinogen in human brains can be used to investigate neurovascular and neuroimmune mechanisms driving neurodegeneration and cognitive decline in AD and other neurological diseases.

Competing Interests: K.A. is a co-founder, scientific advisor, and board director of Therini Bio. Her interests are managed in accordance with the Gladstone Institutes’ conflict-of-interest policy. All other authors declare no conflicts of interest.

Publisher's Disclaimer: “This is an Author Accepted Manuscript version of the following chapter: Merlini, Sozmen, Subramanian, Nana, Seeley, Akassoglou, Three-dimensional Imaging of Fibrinogen and Neurovascular Alterations in Alzheimer’s Disease, published in Alzheimer’s Disease, Methods in Molecular Biology edited by Jerold Chun, 2023, Humana Press reproduced with permission of Springer Humana Press. The final authenticated version is available online at: http://dx.doi.org/10.1007/978-1-0716-2655-9_5.”.

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