Result

“Antibody seems to specifically label β-actin. “ - J. Heller, University College London.

Cell Line: Rat dissociated cortical cul-

ture (14 DIV)

Method of validation: Immunocytochemistry

Primary Antibody: STJ96930 Anti-β-actin anti-

body

Secondary Antibody Donkey anti-mouse CF568

(1:250)

Dilution ratio: 1:100

Protocol

Treatment of materials: Cortical cells were fixed with 3.7% PFA for 12 min at 37°C.

Membrane wash: PBS 2x 5 min, gently rocking at RT ;

Quenching: 0.1% NaBH4 in PBS for 7 min, gently rocking at RT ;

Membrane wash: PBS 1x 2 and 3x 5 min, gently rocking at RT ;

Permeabilization: PBS supplemented with 0.1% triton X-100 (PBS-T) for 10 min, gently rocking at RT ;

Membrane wash: PBS 1x 2 and 3x 5 min, gently rocking at RT ;

Blocking: blocking buffer (PBS supplemented with 3% BSA) for 30 min, gently rocking at RT ;

Primary antibody probing: PBS supplemented with 0.5% BSA overnight at 4°C ;

Membrane wash: PBS 1x 2 and 3x 5 min, gently rocking at RT ;

Secondary antibody probing: Fuorescently-Labelled secondary antibodies diluted in PBS for 1 h, gently rocking at RT;

Membrane wash: PBS 5x 10 min, gently rocking at RT;

Fixation: 4% PFA in PBS for 10 min at RT ;

Membrane wash: PBS 3x 10 min, gently rocking at RT ;

Visualization: Imaging in PBS.

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Fig.1

The antibody labelling showed staining below the plasma membrane as expected. Some cell protrusions are also visualised .