Cell paper, Yang et al (with Cai and Gardinaru, Deisserof former post-doc).
Recently developed clearing techniques
- Clear T, Scale, CUBIC, SeeDB, CLARITY, passive CLARITY, advanced CLARITY, 3DISCO, i3DISCO etc
- this paper, claims first whole organ imaging
- different chemical
- CLARITY: hydrogel and electrophoretic clearing + SDS
- difficult and expensive to electrophoresis. Now have passive version, not ETC
- 3DISCO uses THF/DBE. Faster, better clearing, high quenching of fluorescent proteins.
- iDISCO – immuno labeling. Doesn’t suffer from FP quenching
- passive clearing
- index matched media
- screen different acrylamide and PFA and SDS for optimal concentrations
- like 4% acrylamide + 0% PFA (brain expands a lot but is much clearer). increased detergent concentration.
- also do FISH. much lower cellular background.
- ‘RIMS’ reduce refractive index changes throughout the tissue. Change histodenz concentration
- ‘PARS’ perfusion assisted (similar to cardiac perfusion fixation). Increases speed of clearing.
- much less swelling (probably due to spatial confinement in body?)
- do acralymide and fixation, and clearing during perfusion flow. (after prefusion fixation(?))
- rockefeller lab (also developer of 3DISCO)
- also published in Cell last week
- fix with 4% PFA
- permiablize with methanol
- tissue clearing THF/DCM/BM (DISCO)
- image in DBE
- imaged with Light-sheet microscopy