Matt:LabNotes/2016-7-19
Jump to navigation
Jump to search
CLARITY and PACT for FISSEQ in Mouse Embryo E7.5
- Need more permeabilization to get enzymes and reagents deeper into tissue
- To permeabilize will remove lipids with detergent in long incubation
- Need to embed tissue in hydrogel matrix to create structure that will survive detergent
Experiment
PACT Protocol
- Fix embryo in 4% PFA at 37C for 15min
- Make 4mL PACT solution on ice
- 4% acrylamide in 1X PBS plus 0.25% VA-044 (degassed)
- 3.584mL MilliQ H2O + 0.448mL 10X PBS + 0.448mL 40% acrylamide (cold) + 0.0112g 0.25% VA-044 (cold)
- Wash with 1X PBS
- Incubate embryo in solution at 4C overnight (multiple days?)
- Polymerize gel by incubating at 37C for 3hr
- Make 500mL 8% SDS solution
- 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
- Remove embryo from gel
- Roll on kimwipe to remove bits of gel
- Incubate in 8% SDS at 37C on rotator for overnight (multiple days?)
- Tissue will not be fully clear until put into refractive index matching media
- Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator (2 days?)
- Need to wash away SDS or it will precipitate forming cloudy spots
CLARITY Protocol
- Make 1mL CLARITY solution on ice
- 4% PFA, 0.95% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044, 1X PBS (degassed)
- 4mL 40% PFA + 1mL 40% 19:1 acrylamide:bis + 0.1g VA-044 + 4mL 10X PBS + 31mL H2O
- Can vary the acrylamide/bis-acrylamide concentrations if multiple embryos
- Incubate embryo in solution at 4C overnight
- Polymerize gel by incubating at 37C for 3hr
- Make 500mL 8% SDS solution
- 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
- Remove embryo from gel
- Roll on kimwipe to remove bits of gel
- Incubate in 8% SDS at 37C on rotator for overnight (multiple days?)
- Tissue will not be fully clear until put into refractive index matching media
- Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator (2 days?)
- Need to wash away SDS or it will precipitate forming cloudy spots