Anti-INCENP antibody [58-217] (ab23956)

Immunocytochemistry/ Immunofluorescence - INCENP antibody [58-217] (ab23956)

ICC/IF image of ab23956 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab23956, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunofluorescence - INCENP antibody [58-217] (ab23956)

ab23956 staining INCENP in HeLa cells,  by immunofluorescence.

optimal antibody dilution: 1mg/ml

optimal fixation protocol: PFA/Triton fixation: 10 min room temperature in 3,7 % PFA diluted in PHEM PEM buffer: 45 mM Hepes pH 6,9, 45 mM Pipes pH 6,9, 5mM MgCl2, 10 mM EGTA) containing 0 ,2% Triton X-100, followed  by 3 washes in PBS.  Also gives a good staining with PFA fixation: 10 min in 3,7 % PFA  at room temperature, 5 min in PBS / 0,1%Triton X-100,  then 3 washes in PBS.

IF staining was performed following a standard protocol: blocking: 30 min; primary antibody: 1 hr; secondary antibody: 45’. All incubations were at 37 °C in PBS/ 0,1% Tween containing 3% BSA.

This antibody was also tested using NIH-3T3 cells but it did not provide good results with these cells.

Image kindly submitted by Serena Orlando, Giulia Guarguaglini and Patrizia Lavia, from the University 'La Sapienza' CNR, Italy

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - INCENP antibody [58-217] (ab23956)

ab23956 staining human anal cancer tissue sections by IHC-P.  Sections were formaldehyde fixed and subjected to heat mediated antigen retreival in citrate buffer (pH 6) prior to blocking in a commercially available blocking agent for 20 minutes at 20°C.  The primary anibody was diluted 1/200 and incubated with the sample for 40 minutes at 20°C.  A HRP-conjugated goat anti-mouse antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Antibody Solutions Ltd.

Immunocytochemistry/ Immunofluorescence - INCENP antibody [58-217] (ab23956)

ab23956 staining INCENP in siRNA-treated HeLa cells by Immunocytochemistry/ Immunofluorescence.

Image from Mackay DR et al, J Cell Biol. 2010 Nov 29;191(5):923-31. Epub 2010 Nov 22, Fig 3. DOI 10.1083/jcb.201007124