Double immunofluorescence

Sample preparation

 

  1. sacrifice the animals and dissect knee samples
  2. fix in 95%EtOH 5%HAc over 48h in the fridge
  3. decalcify in 20% EDTApH7.4 for 2 weeks (shaking)
  4. wax embed and section (6um sections)

 

Notes

 

  • this experiment should be repeated on matched sections from 3 unrelated animals per genotype
  • Alexa Fluor® dye spectra:

 

 

 

 

Staining steps

 

dewax in xylene 2 x 5min

 .            ↓

 100% EtOH 3min

 .            ↓

 90% EtOH 3min

 .            ↓

 70% EtOH 3min

 .            ↓

 50% EtOH 3min

 .            ↓

 dH2O 2x 3min (from here on, do not let the samples dry!)

 .            ↓

 1x PBS 2x 3min

 .            ↓

 mark the area around the sections with ImmEdge pen

 keep the slides in a darkened humidified chamber during all incubations

 .            ↓

 2mg/ml bovine hyaluronidase in 1x PBS (antigen unmasking) 45min at 37ºC

 .            ↓

 1x PBS 3x 5min

 .            ↓

 0.5% TritonX 5min

 .            ↓

 1x PBS 2x 5min

 .            ↓

 5ug/ml proteinase K 5min

 .            ↓

 1x PBS 3x 5min

 .            ↓

 block in 10ml PBS/BSA (1% BSA in 1x PBS) + 60ul serum 1h

 .            ↓

 apply mixture of 1ºAb in PBS/BSA 1-18h

 .            ↓

 PBS/BSA 2x 5min

 .            ↓

 apply mixture of 2ºAb in PBS/BSA/serum 1h. 

 .            ↓

 1x PBS 3x 5min

 .            ↓
mount in Vectashield with DAPI
.            ↓
coverslip, store in the dark at 4ºC

 

Data analysis

image the samples and analyse using ImageJ software