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® spectra:

Staining steps

dewax in xylene 2 x 5min

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100% EtOH 3min

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90% EtOH 3min

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70% EtOH 3min

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50% EtOH 3min

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

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1x PBS 2x 3min

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mark the area around the sections with ImmEdge pen

keep the slides in a darkened humidified chamber during all incubations

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0.2% bovine hyaluronidase in 1x PBS (antigen unmasking) 30min or 15 min at 37ºC

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1x PBS 3x 5min

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

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1ºAb in PBS/BSA 1h

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PBS/BSA 2x 5min

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2ºAb in PBS/BSA/serum 1h

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1x PBS 3x 5min

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mount in Vectashield (Vector Labs) with DAPI

Modifications

other unmasking methods:

• intracellular: proteinase K (20 ug/ml in PBS; 37ºC 10min)
• intracellular and TUNEL/DNA fragmentation: citrate buffer boil (10mM citric acid, pH 6.0, 0.05% Tween; microwave on high power 3-4min, medium power 3-4 min, low power 3-4min; allow to cool down on bench)
• intra- and extracellular: 2mg/ml hyaluronidase 45min 37°C, 0.5% Triton X 5min RT,  5μg/ml proteinase K 5min RT (PBS washes in between)

Data analysis

image the samples and analyse using ImageJ software