Free of acrylamide sodium dodecyl sulphate (SDS)‐based tissue clearing (FASTClear): a novel protocol of tissue clearing for three‐dimensional visualization of human brain tissues
In recent years, advances in laser microscopy and endogenous fluorescent tagging techniques have led to the development of many tissue‐clearing strategies, which render tissues optically transparent, allowing large blocks of unsectioned tissue to be visualized in three dimensions (3D). CLARITY (Clear Lipid‐exchanged Acrylamide‐hybridized Rigid Imaging/Immunostaining/In situ hybridization‐compatible Tissue hYdrogel) is one of the tissue‐clearing techniques which works by fixation/hybridization of brain tissue using hydrogel cross‐links and subsequent detergent‐based delipidation to turn the tissue transparent 1. Since CLARITY enables molecular probing using immunofluorescence, this technique was deemed suitable for post mortem human brain tissues to demonstrate the potential in visualizing pathologies in Alzheimer's 2, Parkinson's 3 and neurodevelopmental disorders 1 in 3D. Early attempts using CLARITY were performed on cortical tissue from the brains of children 1, which is considered to be less myelinated than adults, or on thinly sectioned tissues of up to 500 μm in thickness 1. In our previous study, we have demonstrated that CLARITY can successfully render larger blocks of tissue (about 3 mm in thickness) optically transparent in multiple cortical and subcortical regions of the human brain 3. However, we noticed the speed of tissue clearing differs between regions depending on the degree of myelination, and duration of formalin fixation. Densely myelinated regions such as the brainstem and spinal cord in archival formalin‐fixed tissues could not be rendered transparent with CLARITY. Also we, and other groups, have reported tissue expansion after tissue clearing with CLARITY 5. Although it was often claimed that the tissue expansion is a transient effect which will be adjusted by subsequent refractive index matching 6, the effects in human brain tissues, especially after prolonged (>40 days) passive tissue clearing, appeared to be irreversible 3. In addition, immunolabelling with antibodies, particularly on larger samples, remains challenging because the depth of antibody penetration is still limited 7. Many current existing tissue‐clearing protocols are now available and attempts have been made to improve tissue clearing by combining various protocols, such as CUBIC with RIMS in CLARITY and FRUIT (SeeDB with Scale) 8. Our aim was to develop an improved and simplified protocol for tissue clearing in the human brain.
The use of acrylamide hydrogel in the CLARITY protocol poses a number of problems including those described above. Tissues embedded with acrylamide hydrogel undergo expansion upon sodium dodecyl sulphate (SDS) clearing and become more fragile as structural integrity is lost 3. In addition, when transcardial perfusion cannot be performed, diffusion of hydrogel monomers within a large block of tissue may be limited, leading to incomplete tissue hydrogel hybridization 9. Furthermore, although pores in polyacrylamide matrices aid lipid exchange and antibody penetration can be enhanced by changing composition of the hydrogel 5, immunolabelling distance appeared to be better in formaldehyde‐fixed, unhybridized tissue (without acrylamide hydrogel) compared with those hybridized with both formaldehyde and acrylamide 6. The original CLARITY study suggested cross‐links between hydrogel and formaldehyde aid fixation of protein and nucleic acids during the delipidation process. Protein loss was reported to be significantly lower in acrylamide‐embedded tissues compared with unhybridized tissue after SDS clearing 1. However, there has been no evidence showing the existence of hydrogel–formaldehyde cross‐links. Also, in our recently published study, no significant protein loss was found after SDS clearing of formalin‐fixed human brain tissues 10. Besides, protein loss does not necessarily compromise the quality of immunostaining. Hence, as long as the tissue is well fixed in formaldehyde, we recommend the tissue‐clearing procedure to be simplified by omitting the use of acrylamide‐based hydrogel.
Prolonged formaldehyde fixation impedes clearing speed and immunolabelling due to excessive formaldehyde cross‐links on the tissue.