A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Summary

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Abstract

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Introduction

Several tissue clearing techniques have been validated for the use in the brain: hydrogel, hydrophilic, and hydrophobic. These techniques aim to turn a tissue transparent through delipidation, decolorization, and decalcification via the administration of solvents. Once the refractive index of the tissue sample matches the refractive index of the chosen imaging medium, a clear image of the sample can be obtained. Hydrogel based techniques, such as CLARITY, secure biomolecules in the tissue by linking them to acryl-based hydrogels, which prevents structural damage and loss of proteins¹. However, hydrogel techniques utilize harsh chemicals than have the potential to damage more fragile tissues, and denser tissue samples may not be compatible with hydrogel protocol. Certain hydrogel techniques may require expensive equipment or lead to tissue expansion. Hydrophilic techniques, like CUBIC, preserve 3D structure through the formation of hydrogen bonds within the tissue². Tissue expansion can also occur in certain hydrophilic protocol. The clearing ability of hydrophilic techniques often does not match the ability that hydrophobic techniques have, which is important for denser and thicker tissues³.

Hydrophobic techniques are usually fast, do not require special equipment, and produce a sample that is easy to handle and store. iDISCO is a hydrophobic technique that eliminates the shrinkage that can occur in other hydrophobic protocol. Originally, the iDISCO tissue clearing technique was described by Renier et al.⁴ for the embryos and dense, adult organs of mice. This technique removes water from the tissue in the initial dehydration step, thereby reducing the light scatter. Tissue is permeabilized during pretreatment to allow deep antibody penetration. Alexa Fluor dyes in the far-red spectrum are used for immunolabeling to avoid autofluorescence of the tissue at lower wavelengths⁵. Tissue that has undergone hydrophobic clearing protocols are able to be handled easily and can be imaged several times due to the longevity of the Alexa Fluor dyes. If properly stored, tissues can provide images for months to a year after initial processing.

In the present protocol, tyrosine hydroxylase (TH) antibody, Iba1 antibody, and Cholera Toxin Subunit B (CTB) were used on both male and female control and HIV-1 transgenic (Tg) rat brains. The HIV-1 Tg rat possesses seven of the nine genes that comprise the HIV-1 viral genome, which leads to a non-infectious model of long-term HIV-1 protein exposure^6,7,8. Dopaminergic alterations have previously been demonstrated in the HIV-1 Tg rat, and HIV-1 itself is an inflammatory disease, so the antibody choice was relevant to the experimental design^9,10,11,12. CTB is a tracer that attaches to neurons via ganglioside binding and can be used to trace afferent projections in the brain. CTB has been previously used to study projections from the nucleus accumbens to the substantia nigra area, two areas of the brain heavily involved with dopaminergic pathways^13,14,15.

In this protocol, TH will serve as a marker for dopamine production, and Iba1 will mark activated microglia. The TH antibody used selectively labels a single band at approximately 62 kDa that corresponds to tyrosine hydroxylase. The Iba1 antibody corresponds to the Iba1 carboxy-terminal sequence. Both antibodies were raised in rabbit and were polyclonal. CTB will be used for retrograde tracing of neurons from the nucleus accumbens area to the substantia nigra area. The current protocol also offers a guideline for adjustment of the original iDISCO protocol in two distinct ways: 1) overall reduction of the background staining by changing the serum reagents in the blocking steps, and 2) scaling of the incubation time to be suitable for larger tissue samples. Overall, the present protocol provides continued evidence that hydrophobic clearing techniques are feasible in brain tissues of the rat, have no discernible detrimental interactions with HIV-1 viral proteins, and are compatible with TH antibody, Iba1 antibody, and CTB.

Protocol

All animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of South Carolina.

1. Stock solution preparation

1. Solution 1 (1 L): To 900 mL of deionized H₂O add 100 mL of 10x phosphate buffered saline (PBS) and 2 mL of Triton X-100.
2. Solution 2 (1 L): To 900 mL of deionized H₂O, add 100 mL of 10x PBS, 2mL of Tween-20 and 1 mL of 10 mg/mL Heparin stock solution.
3. Solution 3 (500 mL): To 400 mL of Solution 1, add 11.5 g of glycine and 100 mL of dimethylsulfoxide (DMSO).
4. Solution 4 (50 mL): To 42 mL of Solution 1, add 3 mL of corresponding serum and 5 mL of DMSO.
5. Primary antibody solution (50 mL): To 46 mL of Solution 2, add 2.5 mL of DMSO and 1.5 mL of corresponding serum.
6. Secondary antibody solution (50 mL): To 48.5 mL of Solution 2, add 1.5 mL of corresponding serum.
   NOTE: For Solution 4, primary solution, and secondary solution, use a serum that corresponds with the secondary antibody (e.g., goat, horse, bovine).

2. Sample preparation

NOTE: Perform steps 2.1 through 2.7 in a fume hood due to limit the exposure to PFA.

1. Deeply anesthetize young (3-6 week) rat using sevoflurane; proceed to 2.2 when rat is not responsive and fails to respond to toe and tail pinch.
2. Lay the rat in a supine position and make an incision through the abdominal wall using a pair of Iris scissors.
3. Cut up through the bottom of the rib cage to the collarbone on both sides of the rib cage using Mayo scissors.
4. Affix the sternum and ribs away from the heart and lungs with a hemostat.
5. Pierce the left ventricle with a 23 G needle attached to a perfusion pump and cut the right atrium with Iris scissors.
6. Perform transcardial perfusion with approximately 75 mL of 100 mM (1x) PBS.
7. Continue transcardial perfusion with approximately 100 mL of 4% paraformaldehyde (PFA) buffered in 1x PBS.
8. Remove the brain from the skull using forceps.
9. Place the brain in sagittal position and slice into 4 equal sections (approximately 3 mm in width) using a razor blade and a rat brain matrix.
10. Fix each section in 4% PFA in 1x PBS in a sealed, 5mL plastic tube overnight at 4 °C on a shaker.
11. Continue to fix in 4% PFA in 1x PBS at room temperature (RT) on a shaker for 1 h.
12. Wash each section with 1x PBS at RT for 30 min, 3 times.

3. Dehydration and depigmentation

1. Incubate the sample in a 20% methanol/80% deionized water solution for 1 h at RT.
2. Incubate the sample in a 40% methanol/60% deionized water solution for 1 h at RT.
3. Incubate the sample in a 60% methanol/40% deionized water solution for 1 h at RT.
4. Incubate the sample in an 80% methanol/20% deionized water solution for 1 h at RT.
5. Incubate the sample in 100% methanol for 1 h at RT. Repeat with more 100% methanol, incubating for 1 h at RT.
6. Chill the sample in 100% methanol at 4° C for approximately 10 min.
7. Prepare a 66% dichloromethane (DCM)/33% methanol solution.
8. Incubate the sample overnight in the DCM/methanol solution at RT, with shaking.
9. Wash with methanol at RT for 30 min, 2 times.
10. Chill the sample in 100% methanol at 4° C for approximately 10 min.
11. Bleach in chilled, freshly prepared 5% hydrogen peroxide in methanol overnight at 4° C.
12. Incubate the sample in an 80% methanol/20% deionized water solution for 1 h at RT.
13. Incubate the sample in a 60% methanol/40% deionized water solution for 1 h at RT.
14. Incubate the sample in a 40% methanol/60% deionized water solution for 1 h at RT.
15. Incubate the sample in a 20% methanol/80% deionized water solution for 1 h at RT.
16. Incubate the sample in 1x PBS for 1 h at RT.
17. Wash in Solution 1 for 1 h at RT, 2 times.

4. Cholera Toxin Subunit B labeling

1. Insert needle tip of 1 mL syringe into nucleus accumbens site.
2. Slowly inject 2.5 μL of 1% biotin-CTB over 20 s.
3. Leave the needle tip in place for 1 min and remove slowly over 10 s to prevent leakage.

5. Antibody application

1. Incubate the sample in Solution 3 for 2 days at 37 °C in a water bath.
2. Incubate the sample in Solution 4 for 2 days at 37 °C in a water bath.
3. Incubate with primary antibody for 7 days at 37 °C in a water bath.
4. Wash in Solution 2 for 5 times, incubating 1 h per wash. Store in Solution 2 at RT overnight.
5. Incubate with secondary antibody for 7 days at 37 °C in water bath.
6. Wash in Solution 2 for 5 times, incubating 1 h per wash; store in Solution 2 at RT overnight.
   NOTE: All steps from 5.6 onwards, including final storage, should occur in low light. The sample tubes can be shielded with aluminum foil. The concentrations used for the antibodies in this protocol were: TH at 1:100, Iba1 at 1:200, and the secondary antibody at 1:100. Add additional Solution 2 to steps 5.1-5.6 to ensure tubes are completely filled.

6. Tissue clearing

1. Incubate the sample in a 20% methanol/80% deionized water solution for 1 h at RT.
2. Incubate the sample in a 40% methanol/60% deionized water solution for 1 h at RT.
3. Incubate the sample in a 60% methanol/40% deionized water solution for 1 h at RT.
4. Incubate the sample in an 80% methanol/20% deionized water solution for 1 h at RT.
5. Incubate the sample in 100% methanol for 1 h at RT.
6. Incubate in fresh 100% methanol overnight at RT.
7. Incubate in 66% DCM/33% methanol for 3 h, with shaking, at room temperature.
8. Incubate in dibenzyl ether (DBE) without shaking. Leave the sample in DBE until clear and store in DBE until imaging.

7. Mounting

1. Obtain a 3D printed chamber made of Visijet M3 Crystal resin (templates available on the idisco.info website).
2. Secure the chamber to a microscope slide using an epoxy.
3. Place the sample in the square space in the middle of the chamber.
4. Fill the chamber completely with DBE.
5. Place a 0.17 mm thick coverslip over the chamber and apply pressure until the coverslip has full contact with the chamber walls.
6. Seal the edges of the coverslip to the chamber using the epoxy.
7. Rotate the chamber to allow air bubbles to escape from the filling inlet.
8. Fill the chamber with additional DBE.
9. Seal the filling inlet with epoxy and allow to cure.
   NOTE: Excess DBE will spill out during mounting.

8. Imaging

1. Use a confocal microscope system to perform z-scan at 4x magnification (see the attached 3D video).
2. Achieve fluorescent excitation of the sample using a laser set to emit at a wavelength similar to the wavelength specified by the secondary antibody. TH excitation was achieved utilizing a HeNe laser set to emit at 632 nm.
3. Set the detector to a wavelength close to the laser emission. For the present protocol, the detector for TH was set to 650 nm.
4. Raise the gain of the detector until there is a visible signal. The 650LP gain for images was set to 7.50 B.
5. Using the Scan option in the confocal imaging software, move the microscope stage until the sample is brought into focus.
6. Navigate around the tissue in all plains and obtain either single images or z-stack images using the confocal imaging software.
7. Once imaging is completed, remove the sample from the chamber. Place it back into a tube completely filled with DBE and store at RT in a dark place, preferably covered in aluminum foil.
   NOTE: Samples can be remounted and imaged as long as the florescence signal is still strong.

Results

Full clearing of large sections of both F344/N and HIV-1 rat brain tissue was achieved using this modified hydrophobic tissue clearing protocol. Figure 1 displays a typical confocal image for TH in the substantia nigra region. Figure 1A represents dense, positive staining. Dense areas such as these can be parsed out by focusing through the “Z” plane to confirm positive staining and proper cell morphology. Figure 1B represents sparse...

Discussion

Tissue clearing offers a solution to the limitations of traditional IHC protocol. A sample that is transparent minimizes the scattering and absorption of light, which provides cellular level optical access to intact tissues^18,19. Tissue clearing techniques turn a tissue transparent through delipidation, decolorization, and decalcification with the administration of solvents. Once the refractive index of the tissue sample matches the refractive index of the chosen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cholera Toxin Subunit B (Recombinant), Alexa Fluor 488	Invitrogen	C34775
DBE	Sigma-Aldrich	108014-1KG
DCM	Sigma-Aldrich	270997-100mL
DMSO	Sigma-Aldrich	472301-1L
Glycine	Fisher Chemical	G46-500
Goat anti-rabbit Alexa Fluor Plus 647	Invitrogen	A32733
Goat serum	Sigma Life Science	G9023-10mL
Heparin	Acros Organics	41121-0010
Iba1 primary antibody	FUJIFILM Wako	019-19741
Kwik-sil epoxy	VWR	70730-062
Methanol	Sigma-Aldrich	34860-1l-R
PBS	Fisher Bioreagents	BP2944-100
Perfusion machine	VWR	70730-062	mini pump variable flow
PFA	Sigma-Aldrich	158127-3KG
TH primary antibody	Millipore Sigma	AB152
TritonX-100	Fisher Bioreagents	BP151-500
Tween-20	Fisher Bioreagents	BP337-500