Immunohistochemistry

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)

Note

 to generate reliable data, this experiment should be performed on matched sections from 3 unrelated animals per genotype

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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 225ml MetOH + 7.5ml H2O2  30min (quench endogenous peroxidase)

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 1x PBS 3x 5min

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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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 prepare ABC reagent (2.5ml PBS+dropA+dropB, 30min before use)

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 1x PBS 3x 5min

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 ABC reagent 30min

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1x PBS 2x 5min

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DAB (prepare fresh) 2-10min, stop in dH2O

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methyle green 10 min

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dunk in 3 fresh changes of tap water

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95% EtOH 3min

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 100% EtOH 5min

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dehydrate in xylene 2x 5min
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coverslip, dry overnight

Modifications

1. if using a fluorescent secondary, skip the peroxidase quenching step and mount in Vectashield straight after washing off the excess of the 2º antibody
2. other unmasking methods: proteinase K (20 ug/ml in PBS; 37ºC 10min), 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)

 end result: positive staining brown, nuclei green