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 (HRP-DAB) system (16 min/ 37°C) before being counterstained with hematoxylin.

ab10558 staining CD45 in Human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Negative control is shown in panel. Blocking was with horse serum (1/75) for 1 hour at room temperature. Samples were incubated with primary antibody (1/10) overnight at 4°C. A Biotin-conjugated Horse anti-mouse polyclonal (1/200) was used as the secondary antibody.

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.

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

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

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size: 147 kDa


Exposure time: 3 minutes

Secondary antibody - goat anti-rabbit H&L (HRP) (ab6721)

IHC image of CD45 antibody staining in a section of formalin-fixed paraffin-embedded normal human spleen* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab10558, 1ug/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-CD45 antibody (ab10558) at 1 µg/ml

Lane 1 : Jurkat (Human) Whole Cell Lysate
Lane 2 : RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate
Lane 3 : Spleen (Mouse) Tissue Lysate
Lane 4 : Spleen (Rat) Tissue Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 147 kDa
Observed band size: 190 kDa why is the actual band size different from the predicted?
Additional bands at: 230 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab10558 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

CD45 contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

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.

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Immunohistochemical analysis of formaldehyde fixed human cephalic sections. Primary antibody ab10558 to CD45 incubated at a concentration of 1/100 for 4°C for 18 hours. Secondary antibody used was a goat anti-rabbit congugated to biotin at a 1/200 dilution. Blocking was done with serum at a 10% concentration for 1 hour at 25°C.

See Abreview