Immunohistochemistry (Frozen sections) - PCNA antibody [PC10] - Proliferation Marker (ab29)

Mouse monoclonal [PC10] to PCNA - Proliferation Marker (ab29) used in immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections;1/6000 for 2h at RT) on intestine of adult Zebra fish). Antigen retrieval step: Heat mediated. Blocking step: 1% BSA for 10 mins at RT. Incubation time: Secondary Antibody: Biotin conjugated goat anti mouse Igs (1/200). NB: The crypt nuclei on this image of zebrafish intestine, are positive for the PCNA/PC10 clone conforming to accepted localisation data for PCNA in other species.

Carl Hobbs, CARD, KCL, UK

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - PCNA antibody [PC10] - Proliferation Marker (ab29)

Mouse monoclonal [PC10] to PCNA - Proliferation Marker (ab29) used in immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections; 1/6000 for 2h at RT) on Human Tissue sections (Paget's disease of the nipple). Antigen retrieval step: Heat mediated. Blocking step: 1% BSA for 10 mins at RT. Incubation time: Secondary Antibody: Biotin conjugated goat anti mouse Igs (1/200).

Carl Hobbs, CARD, KCL, UK

Western blot - PCNA antibody [PC10] - Proliferation Marker (ab29)

All lanes : Anti-PCNA antibody [PC10] - Proliferation Marker (ab29) at 5 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 29 kDa
Observed band size : 29 kDa


Exposure time : 4 minutes

Flow Cytometry - Anti-PCNA antibody [PC10] - Proliferation Marker (ab29)

Overlay histogram showing HeLa cells stained with ab29 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab29, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Western blot - PCNA antibody [PC10] - Proliferation Marker (ab29)

All lanes : Anti-PCNA antibody [PC10] - Proliferation Marker (ab29) at 1/5000 dilution

Lane 1 : Human 293 total cell extract
Lane 2 : Chicken DT40 total cell extract


Performed under reducing conditions.

Predicted band size : 29 kDa
Observed band size : 30 kDa (why is the actual band size different from the predicted?)


Exposure time : 5 seconds

Blocking for 2 hours in 5% milk. Primary antibody incubated for 1 hour.

Taken from an Abreview submitted by Javier di Noia

Immunoprecipitation - PCNA antibody [PC10] - Proliferation Marker (ab29)

ab29 at a 1/500 dilution used in immunoprecipitation.

Lane 1: Beads only + HeLa whole cell lysate

Lane 2: Beads only + SupT1 whole cell lysate

Lane 3: Beads + ab29 + HeLa whole cell lysate

Lane 4: Beads + ab29 + SupT1 whole cell lysate

A 50KDa band is precipitated which is IgG heavy chain whilst the 30kDa band is PCNA.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - PCNA antibody [PC10] - Proliferation Marker (ab29)

ab29 at 1/6000 staining mouse embryo (day 17) liver and gut tissue sections by IHC-P. The tissue was formaldehyde fixed and a heat mediated antigen retrieval step in Tris buffer was performed. The tissue was blocked before incubation with the antibody for 2 hours. A biotinylated goat polyclonal antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Mr Carl hobbs

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - PCNA antibody [PC10] - Proliferation Marker (ab29)

Mouse monoclonal [PC10] to PCNA - Proliferation Marker (ab29) used in immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections; 1/6000 for 2h at RT) on E6 developing chick brain). Antigen retrieval step: Heat mediated. Blocking step: 1% BSA for 10 mins at RT. Incubation time: Secondary Antibody: Biotin conjugated goat anti mouse Igs (1/200). NB: This image shows developing brain/overlying skin.

Carl Hobbs, CARD, KCL, UK

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - PCNA antibody [PC10] - Proliferation Marker (ab29)

Immunohistochemistical staining (Formaldehyde/PFA-fixed paraffin-embedded sections) for PCNA antibody [PC10] - Proliferation Marker (ab29) on Rat Tissue sections (adult spinal cord DRG). Antigen retrieval step: Heat mediated. Blocking step: 1% BSA for 10 mins at RT. Primary Antibody used at 1/6000 for 2 minutes at RT. Secondary Antibody: Biotin labelled goat anti mouse Igs (1/200).

Carl Hobbs, CARD, KCL, London, UK

Immunocytochemistry/ Immunofluorescence - PCNA antibody [PC10] - Proliferation Marker (ab29)

ICC/IF image of ab29 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab29, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HEK 293, HepG2 and MCF7 cells.

Immunohistochemistry (Frozen sections) - PCNA antibody [PC10] - Proliferation Marker (ab29)

ab29 staining PCNA in mouse embryonic brain tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with paraformaldehyde and permeabilized with 0.1% PBS-Tween before blocking with 5% BSA for 1 hour at 22⁰C. The sample was incubated with primary antibody (1/500) in 5% BSA in 0.3% PBS-Triton-X100 for 14 hours at 22⁰C. An Alexa Fluor^®488-conjugated Goat polyclonal to mouse IgG was used as secondary antibody at 1/500 dilution.

This image is a courtesy of Anonymous Abreview

Immunocytochemistry/ Immunofluorescence - PCNA antibody [PC10] - Proliferation Marker (ab29)

ICC/IF image of ab29 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab29, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) 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.