wholemount_for_adipose
Wholemount staining
Work at 4 ˚C at all times!
Day 1
- Dissect the adipose tissue on dry ice, try to slice as thin a sheet of tissue as possible, that will fit in a 96-well plate well.
- Wash 3 times, first wash with 50 mM Glycine 5 min, then 2x 10 min in PBS
- Permeabilise cells and block non-specific binding in 1 mL B1 buffer on rotating platform (1 hour)
- Aspirate the B1 buffer and then incubate tissue with appropriate dilution of primary antibody in B1 buffer on shaking incubator o/n
Day 2
- Wash 3 times with 1 mL B2 buffer, 10 min on rotating platform
- Incubate with appropriate dilution of secondary antibody in B1 buffer and counterstain with Bodipy and DAPI on shaking incubator (o/n) COVER WITH ALU FOIL
Day 3
- Wash 3 times with 1 mL B2 buffer, 10 min on rotating platform for the first two washes and o/n for the last.
- Mount specimen in 80% glycerol/1x PBS
Example Antibody concentrations
| Primary | Secondary |
|---|---|
| Mab3211 (mouse) 1:200 | Alexa 555 1:10000 1) |
| F4/80 (rat) 1:50 | Alexa 647 1:500 |
| Bodipy 1:5000 | |
| DAPI 1:2000 | |
| B1 buffer | 1% BSA, 5% normal goat serum, 0.3% Tween PBST in PBS, keep at 4 ˚C |
| B2 buffer | 0.1% Tween PBST in PBS |
long:18202748
1)
Or even 1:100000 if there is a high background in the red channel
wholemount_for_adipose.txt · Last modified: 2021/01/19 11:10 by 127.0.0.1