Anti-CDKN2A/p14ARF antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CDKN2A/p14ARF antibody (AB3642)

ICC/IF image of ab3642 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab3642 at 1μg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CDKN2A/p14ARF antibody (AB3642)

Confocal image showing nuclear staining increased after HeLa cells transfected with CDKN2A/p14ARF.

ICC/IF image of ab3642 stained HeLa cells. The cells were 4% paraformaldehyde fixed and then incubated in 0.1% trixtonX-100 to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with primary antibody ab3642 at a dilution of 1/250. An AlexaFluor®594 goat anti-mouse secondary IgG (ab150077) was used at a 1/1000 dilution. Anti-tubulin (ab7291) and an AlexaFluor®594 goat anti-mouse IgG (ab150120) were used as counterstains, both at a dilution of 1/1000. DAPI was used to stain the cell nuclei blue.

• IHC-P

AbReview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p14ARF antibody (AB3642)

Immunohistochemical analysis of human cervical adenocarcinomas tissue labelling p14 with ab3642 at 1/200 dilution in PBS/1% BSA. Staining was most prominent in the nuclei but other staining, most likely aspecific staining, was observed in apical vacuoles.

Dilutions of 1/100, 1/200 and 1/400 were tested.
1/100 dilution was a bit too weak and 1/400 gave too much background so in the end the dilution of 1/200 in PBS containing 1% BSA was used.

Several heat induced antigen retrieval methods were used (citrate and EDTA-Tris, both in microwave and autoclave).

Image supplied as part of the review submitted by Joost van Galen.