The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 1µg for 106 cells. Also see PubMed: 21247888
ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
1/5 - 1/20.
1/5 - 1/20. Formalin fixation and embedding in paraffin may affect the reactivity of the antibody.
1/5 - 1/20.
RelevanceA (pre)melanosomal 100 + 7 kd antigen (glycoprotein) present in melanomas, clear cells sarcomas (melanoma of soft tissue), nevocellular nevi, and normal melanocytes. Except for one case of non Hodgkin's lymphoma in which macrophages were positive, the antigen has not been detected with other tumors or tissues observed.
Cellular localizationThe innerside of membranes of cytoplasmic vesicles in melanoma cells.
ab34165 staining Melanoma Associated Antigen 100+ / 7 kDa in LG2-MEL-220 (Mel220), a human Pmel17-deficient melanoma cell line by Immunocytochemistry/ Immunofluorescence. Mel220 cells were transfected with wild type Pmel17 and IR-wt mutant Pmel17. The cells were seeded on glass coverslips and washed with PBS containing 0.9 mm CaCl2 and 0.5 mm MgCl2 and then fixed with 2% formaldehyde for 15 min at room temperature. After quenching with PBS containing 10 mm glycine followed by a wash with PBS/0.5% bovine serum albumin, cells were permeabilized for 1h in staining buffer (PBS/0.5% bovine serum albumin/0.5% saponin). The primary antibody was diluted 1/20 and incubated with sample for 1 hour. An Alexa Fluor®647 conjugated anti-mouse secondary was used at 1/100 dilution. anti-HMB50 was from another source.
IHC image of ab34165 staining in human melanoma 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 ab34165 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.
Overlay histogram showing Malme-3 cells stained with ab34165 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab34165, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Malme-3 cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.