All lanes : Anti-PCNA antibody [PC10] (ab29) at 1 µg/ml

Lane 1 : HeLa 20 ug
Lane 2 : PC12 20 ug
Lane 3 : SV40LT-SMC 20 ug
Lane 4 : NIH 3T3 20 ug
Lane 5 : Rat liver 20 ug
Lane 6 : Rat heart 20 ug

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/20000 dilution

Performed under reducing conditions.

Predicted band size: 29 kDa

Merged signal (red and green). Green - ab29 observed at 32 kDa. Red - loading control, ab52866, observed at 50 kDa.

All samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab29 and ab52866(Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1 ug/mL and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

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).

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All lanes : Anti-PCNA antibody [PC10] (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
All lanes : 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

ab29 stained in HeLa cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab29 at 5 µg/ml and ab6046 (Rabbit polyclonal to beta tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150117 (pseudo-colored red) and ab150080 (colored green) used at 1 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

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 20 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, 0.1μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

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.

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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).

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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.

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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 zebrafish). 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.

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IHC image of PCNA staining in rat large intestine formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab29, 0.025µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

IHC image of PCNA staining in rat spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab29, 1/30,000 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Aldehyde dehydrogenase (ALDH1) expression correlates with clinical outcome of breast cancer patients

(A) Immunohistochemical staining shows tumors with poor clinical response (progressive or stable disease, PD/SD) to neo-adjuvant chemotherapy express high ALDH1 (>20% positive cancer cells) in pre-chemotherapy samples, and tumors with partal response (PR) express low ALDH1 (≤20% positive cancer cells). High proliferating cell nuclear antigen (PCNA)(>25% positive cancer cells) and poor apoptosis are observed in tumors with PD/SD after neo-adjuvant chemotherapy. Representative images of ALDH1 (×200), PCNA (×200) and Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP labeling (TUNEL,×400).

PCNA is detected using ab29 at 1/100 dilution.

(From Figure 1A of Gong et al)

Annexin V and PCNA staining of decellularized lung scaffolds recellularized with (A) MSCs in static versus bioreactor conditions for 14 (panels A, B for annexin V, and a, b for PCNA) and 28 days (panels C, D for annexin V, and c, d for PCNA) (single cells), (B) MSC cell clusters in static conditions at 14 days (panel A for annexin V, and a for PCNA), (C) C10 cells in static (panel A for annexin V, a for PCNA) versus bioreactor (panel B for annexin V, b for PCNA) conditions for 11 days.

An inset in Fig 4Ab with higher magnification is shown to demonstrate that a majority of the cells stained positive for PCNA. Cell nuclei are labeled in blue; marker of interest is labeled in green. Magnifications are 400x. Overlap of cell nucleus and marker of interest can generate green or white color. For each condition, images are representative of the entire lung.

MSCs = Bone marrow-derived mouse mesenchymal stromal (stem) cells.

PCNA is detected using ab29 at 1/1000 dilution.

(From Figure 4 of Crabbe et al)