Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [PAb 240] (ab26)

IHC image of ab26 staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab26, 5µg/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.

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [PAb 240] (ab26)

ICC/IF image of ab26 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 ab26 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) 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.

Western blot - Anti-p53 antibody [PAb 240] (ab26)

All lanes : Anti-p53 antibody [PAb 240] (ab26) at 5 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Hela Whole Cell Lysate - Bleomycin Treated (40U/ml)

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 53 kDa


Exposure time : 4 minutes

Western blot - p53 antibody [PAb 240] (ab26)

Anti-p53 antibody [PAb 240] (ab26) at 1/2000 dilution + Human HEK293 whole cell lysate.

Performed under reducing conditions.

Predicted band size : 53 kDa
Observed band size : 50 kDa (why is the actual band size different from the predicted?)
Additional bands at : 75 kDa (possible cross reactivity).

Dr. Jianping Ye, MD, Louisiana State University

Western blot - p53 antibody [PAb 240] (ab26)

All lanes : Anti-p53 antibody [PAb 240] (ab26) at 1/2000 dilution

Lane 1 : Human breast cancer cell-line, MCF7 cells (p53 WT), whole cell lysate
Lane 2 : Human breast cancer cell-line, MDA231 cells (p53 Mutant), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
HRP conjugated donkey anti-mouse antibody at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 53 kDa
Observed band size : 53 kDa
Additional bands at : 72 kDa (possible non-specific binding).

Exposure time : 10 seconds

This image is courtesy of an Abreview submitted by Dr Cherie Blenkiron

Immunocytochemistry/ Immunofluorescence - p53 antibody [PAb 240] (ab26)

ab26 staining p53 in rat bone marrow cells by Immunocytochemistry/ Immunofluorescence. The cells were formaldehyde fixed, permeabilised in 0.1% Triton X-100 and then blocked using 5% BSA for 1 hour at 25ºC. Samples were then incubated with primary antibody at 1/250 for 9 hours at 4ºC. The secondary antibody used was a goat anti-mouse IgG conjugated to Alexa Fluor® 594 (pink) used at a 1/250 dilution. DAPI was used to stain the cell nuclei (blue).

This image is a courtesy of Anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - p53 antibody [PAb 240] (ab26)

ab26 staining p53 in Mouse bone tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% serum for 1 hour at 20ºC; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/400) for 12 hours at 4ºC. A Biotin-conjugated Rabbit anti-mouse polyclonal (1/200) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - p53 antibody [PAb 240] (ab26)

ab23106 staining p53 in Mouse bone marrow cells by Immunocytochemistry/ Immunoflourescence. Cells were fixed in formaldehyde and permeabilized in 0.1% Triton X-100 prior to blocking in 5% BSA for 1 hour at 25ºC. The primary antibody was diluted 1/250 in PBS and incubated with the sample for 9 hours at 4ºC. The secondary antibody was Alexa Fluor® 594-conjugated Goat anti-Mouse polyclonal, diluted 1/500.
Nuclei were counterstained blue with DAPI.

This image is courtesy of an Anonymous Abreview.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [PAb 240] (ab26)

ab26 staining p53 in canine gastric adenocarcinoma tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

Tissue was fixed in formaldehyde and a heat mediated antigen retrieval step was performed using citrate. Samples were then incubated with ab26 at a 1/200 dilution for 12 hours at 4°C. The secondary used was an undiluted biotin conjugated polyclonal.

This image is courtesy of Dr Alejandro Suarez by Abreview.