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 ha....

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 ser....

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