The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml.
Use a concentration of 2.5 µg/ml. Detects a band of approximately 55 kDa (predicted molecular weight: 66 kDa).Can be blocked with Human Glypican 3 peptide (ab111665). Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.
Cell surface proteoglycan that bears heparan sulfate. Inhibits the dipeptidyl peptidase activity of DPP4. May be involved in the suppression/modulation of growth in the predominantly mesodermal tissues and organs. May play a role in the modulation of IGF2 interactions with its receptor and thereby modulate its function. May regulate growth and tumor predisposition.
Highly expressed in lung, liver and kidney.
Involvement in disease
Defects in GPC3 are the cause of Simpson-Golabi-Behmel syndrome type 1 (SGBS1) [MIM:312870]; also known as Simpson dysmorphia syndrome (SDYS). SGBS is a condition characterized by pre- and postnatal overgrowth (gigantism) with visceral and skeletal anomalies.
ICC/IF image of ab66596 stained Hek293 cells. The cells were 100% methanol fixed (5 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 (ab66596, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.
IHC image of ab66596 staining in human placenta 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 ab66596, 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.