Immunocytochemistry

Sample preparation

seed cells in 8well glass bottomed chamber slides (Nunc) at 10-30,000 cell per well

grow until confluent

Protocol

fix cells in 4%PFA for 15 min

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wash twice in ice cold 1x PBS

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store in the fridge overnight (in PBS, wrapped in clingfilm)

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permeabilise cells with PBS containing 0.25% Triton X-100 for 10 min (intracellular proteins)

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wash 3 x 5min in PBS

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block with 10% Donkey serum in PBS for 30 min at room temperature

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incubate with diluted 1° antibodies in 10% donkey serum in PBS for 1 h at room temperature

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wash 3 x 5min in PBS

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incubate with 2° antibodies in 10% donkey serum in PBS for 1 hr at room temperature

(for fluorescent ICC limit the light exposure!)

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wash 3 x 5min in PBS

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mountin Vectashield with DAPI and cover slip

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image and analyse using ImageJ software