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wholemount_for_adipose

Wholemount staining

Work at 4 ˚C at all times!

Day 1

  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.
  2. Wash 3 times, first wash with 50 mM Glycine 5 min, then 2x 10 min in PBS
  3. Permeabilise cells and block non-specific binding in 1 mL B1 buffer on rotating platform (1 hour)
  4. Aspirate the B1 buffer and then incubate tissue with appropriate dilution of primary antibody in B1 buffer on shaking incubator o/n

Day 2

  1. Wash 3 times with 1 mL B2 buffer, 10 min on rotating platform
  2. 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

  1. Wash 3 times with 1 mL B2 buffer, 10 min on rotating platform for the first two washes and o/n for the last.
  2. Mount specimen in 80% glycerol/1x PBS

Example Antibody concentrations

PrimarySecondary
Mab3211 (mouse) 1:200Alexa 555 1:10000 1)
F4/80 (rat) 1:50Alexa 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

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1)
Or even 1:100000 if there is a high background in the red channel
wholemount_for_adipose.txt · Last modified: 2014/08/15 14:17 (external edit)