The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/1000. Detects a band of approximately 40 kDa (predicted molecular weight: 34 kDa).
Receptor for TNFSF5/CD40LG.
B-cells and in primary carcinomas.
Involvement in disease
Defects in CD40 are the cause of hyper-IgM immunodeficiency syndrome type 3 (HIGM3) [MIM:606843]; also known as hyper-IgM syndrome 3. HIGM3 is an autosomal recessive disorder which includes an inability of B cells to undergo isotype switching, one of the final differentiation steps in the humoral immune system, an inability to mount an antibody-specific immune response, and a lack of germinal center formation.
IHC image of CD40 staining in Human normal tonsil 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 ab13545, 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.
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.
Anti-CD40 antibody (ab13545) at 1/1000 dilution + Recombinant human CD40 protein (Fc Chimera Active) (ab83917) at 1 µg
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 34 kDa
Exposure time : 8 minutes
Western blot - Anti-CD40 antibody (ab13545)
All lanes : Anti-CD40 antibody (ab13545) at 1/200 dilution
Lane 1 : ESK4 cell lysate Lane 2 : Molt 4 cell lysate
Predicted band size : 34 kDa
Western blot - CD40 antibody (ab13545)
All lanes : Anti-CD40 antibody (ab13545) at 1/1000 dilution
Lane 1 : Control human HCAEC cells - whole cell lysate Lane 2 : Control human HCAEC cells - whole cell lysate Lane 3 : Treated human HCAEC cells - whole cell lysate Lane 4 : Treated human HCAEC cells - whole cell lysate
Secondary HRP conjugated Goat Anti-Rabbit IgG Developed using the ECL technique
Western blot - CD40 antibody (ab13545)This image is courtesy of an anonymous Abreview
All lanes : Anti-CD40 antibody (ab13545) at 1/5000 dilution
Lane 1 : Human B chronic lymphocytic leukaemia cells, nuclear fraction Lane 2 : Human B chronic lymphocytic leukaemia cells, cytoskeletal fraction Lane 3 : Human B chronic lymphocytic leukaemia cells, membrane fraction Lane 4 : Human B chronic lymphocytic leukaemia cells, cytosolic fraction
Lysates/proteins at 18 µg per lane.
Secondary HRP conjugated goat anti-rabbit Developed using the ECL technique
Shoji T et al. Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models. Neuro Oncol18:1120-8 (2016).
Read more (PubMed: 26917236) »
Qiu X et al. CD40 ligand induces RIP1-dependent, necroptosis-like cell death in low-grade serous but not serous borderline ovarian tumor cells. Cell Death Dis6:e1864 (2015).
Read more (PubMed: 26313915) »