Alistair's Lab Notebook

advanced CARITY
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

Differences

• 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

Advanced CLARITY

• passive clearing
• index matched media

Optimization

• 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(?))

iDISCO technique

Paper info

• rockefeller lab (also developer of 3DISCO)
• also published in Cell last week

method

• fix with 4% PFA
• permiablize with methanol
• immunolabel
• tissue clearing THF/DCM/BM (DISCO)
• image in DBE
• imaged with Light-sheet microscopy