Result

“Antibody seems to specifically label βIII-tubulin, highlighting neurites of the cultured cells.“ - J. Heller, University Col-lege London.

Cell Line: Rat dissociated cortical cul-

ture (14 DIV)

Method of validation: Immunocytochemistry

Primary Antibody: STJ97123 Anti-β III Tubulin

antibody

Secondary Antibody Goat anti-rabbit AlexaFluor

647 (1:500)

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 reveals neurites of cultured cells.