Western blot - Anti-CD45 antibody (ab10558)

All lanes : Anti-CD45 antibody (ab10558) at 1/500 dilution

Lane 1 : Jurkat Whole Cell Lysate
Lane 2 : Jurkat Whole Cell Lysate with CD45 peptide (ab17553)

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size : 147 kDa


Exposure time : 3 minutes

Flow Cytometry - Anti-CD45 antibody (ab10558)

Asynchronous KM-H2 cells were pelleted and labeled by indirect immunofluorescence. Cells were stained with ab10558 (1/200) for 30min at 4'C, washed and then stained with goat anti-rabbit alexafluor 488 (1/200). Forward/Side scatter were used to eliminate cellular debris. The accompanying marker was applied such that only 2% of the IgG control was positive Based on the accompanying image, approximately 8.4% of cells exhibited positive staining for anti-CD45. Since KM-H2 are known to have low levels of CD45 transcripts they are expected to have low levels of CD45, which is reflected in the ~8%. This image is from an Abreview.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - CD45 antibody (ab10558)

ab10558 (1:40) staining CD45 in paraffin-embedded human tonsil (left panel) using an automated system (Ventana Discovery). Right-hand panel shows negative control (no primary antibody).
Using this protocol there is strong membrane staining of B cells in the germinal centres and mantle zone of the follicles and scattered cells of the interfollicular areas (paracortical T and B cells). There is a mild to moderate degree of cytoplasmic staining associated with the membrane staining in these specific cells.

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Extended Retrieval programme. Slides were blocked in 3% H₂O₂ /4 min/ 37°C and incubated with ab10558 (1:40 dilution / 1 hour/ 37°C). Sections then blocked (4mins/ 37°C) and incubated with Dako swine anti-rabbit antibody (1:50, 28 min/ 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC system (16 min/ 37°C) and Ventana DAB map reagent (8 min/ 37°C). Slides were counterstained with Haematoxylin and coverslipped in DPX.

For manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunohistochemistry (Frozen sections) - CD45 antibody (ab10558)

ab10558 staining CD45 in fetal mouse liver tissue sections 12.5 dpc by Immunohistochemistry (frozen sections). Tissue was fixed with paraformaldehyde and a permeabilization step was performed using 0.1% Tween-20. Samples were then blocked with 5% BSA for 1.5 hours at 20ºC followed by incubation with the primary antibody at a 1/150 dilution, for 16 hours at 4ºC. A Goat anti-rabbit Alexa Fluor® 594 polyclonal was used as the secondary antibody at a 1/1500 diution.

Image courtesy of Mr Sam Lee by Abreview.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - CD45 antibody (ab10558)

IHC image of CD45 staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 ab10558, 1µg/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.

Flow Cytometry - Anti-CD45 antibody (ab10558)

Overlay histogram showing Jurkat cells stained with ab10558 (red line). The cells were fixed with 80% methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab10558, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/1000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.