What is iDISCO?
iDISCO is a method developed by Renier and co., whereby immunolabelling is combined with a method which effectively makes biological tissues transparent (3DISCO). This means that you are not only able to label tissue, but also you are able to see labels within a 3D block of tissue. Complex structures, such as axonal tracts, cannot easily be determined by 2D imaging, therefore 3D imaging is preferable to view such structures.
Visualizing FOXP2 in Mouse Brain
FOXP2 is a transcription factor involved in vocalization, and is present throughout the brain. Transcription factors are proteins which help the transcription of DNA into mRNA, which in turn gets translated into proteins. Renier and co. used iDISCO to see if FOXP2 could be detected in mouse brains, and they found that FOXP2 could be seen deep within the brain. This confirmed the findings of other researchers, who used different methods.
Visualizing Axonal Projections
Axons are projections from neurons (nerve cells) which carry nerve impulses away from the neuron to neighboring neurons or muscles. Due to the range that nerve cells need to cover, these axons can be extremely long. Renier and co. used antibodies that targeted specific neurons in mouse embryos and brains, and showed that iDISCO made it possible to visualize these neurons in mouse embryo heads without dissection. It was also possible to visualize neurons and their projections within certain parts of the adult mouse brain. This technique was also used to successfully visualize other types of neurons.
Does iDISCO Work with Transgenic Markers?
Antibodies cannot be used to visualize all types of structures, therefore Renier and co. set about to see if markers expressed by transgenic mice can be visualized using iDISCO. Several techniques, including the use of fluorescent markers and epitope tags like LacZ showed that iDISCO can be used. This included the visualization of specific types of neurons, and using an antibody to detect the LacZ product to visualize cells producing this marker.
Studying Alzheimer’s Disease Using iDISCO
Alzheimer’s disease is a neurodegenerative disorder characterized by two hallmark changes within the brain: the accumulation of beta amyloid (β-amyloid) proteins leading to it aggregating within the brain (β-amyloid plaques), and the presence of intracellular neurofibrillary tangles (NFTs). It is still unclear how these hallmarks come to form, therefore the study by Liebmann set about to see if iDISCO can be utilized for this purpose.
Liebmann and co. first set about to see if β-amyloid plaques in mouse brains could be visualized by iDISCO. Initially, they used antibodies but then used Congo red, a small molecule known to bind to β-amyloid plaques. They found that antibodies and Congo red can be successfully used to visualize β-amyloid plaques in mouse brains. Using Congo red has the advantage that it only needs 2 hours before the dye is visible by iDISCO, which is much quicker than using antibodies.
Liebmann and co. verified that iDISCO gave a similar number of β-amyloid plaques when compared with a standardized technique for visualizing β-amyloid plaques known as immunohistochemistry, therefore iDISCO is as good as immunohistochemistry for detecting β-amyloid plaques.
The advantage of iDISCO is that large volumes of intact tissue can be visualized while maintaining good resolution of structures. The structures formed by β-amyloid plaques can be clearly seen using iDISCO. Therefore, iDISCO can be used to determine different structures formed by β-amyloid plaques.
Marking Immediate Early Genes to Study Brain Activity
Immediate Early Genes are genes which are switched on rapidly, and do not require the involvement of other proteins. Some Immediate Early Genes, such as c-Fos, ARC, EGR1, FOSB and NPAS4, have been used in the past as a way to determine brain activity. Therefore, Ranier and co. specifically labelled Immediate Early Genes and used the iDISCO approach to visualize the activity of Immediate Early Genes.
In this study, Ranier and co. modified the iDISCO so that the sample did not shrink, and this means that the image produced by the modified iDISCO, or iDISCO+, can be compared to a reference.