Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. Probably involved in vesicular trafficking via its association with the CART complex. The CART complex is necessary for efficient transferrin receptor recycling but not for EGFR degradation.
Involvement in disease
Defects in ACTN4 are the cause of focal segmental glomerulosclerosis type 1 (FSGS1) [MIM:603278]. A renal pathology defined by the presence of segmental sclerosis in glomeruli and resulting in proteinuria, reduced glomerular filtration rate and edema. Renal insufficiency often progresses to end-stage renal disease, a highly morbid state requiring either dialysis therapy or kidney transplantation.
Nucleus. Cytoplasm. Localized in cytoplasmic mRNP granules containing untranslated mRNAs. Colocalizes with actin stress fibers. Nuclear translocation can be induced by the PI3 kinase inhibitor wortmannin or by cytochalasin D. Exclusively localized in the nucleus in a limited number of cell lines.
Immunocytochemistry/ Immunofluorescence - Anti-alpha Actinin 4 antibody (ab59468)This image is courtesy of an anonymous Abreview
ab59468 staining alpha Actinin 4 from mouse embryonic fibroblasts by immunocytochemistry/ immunofluorescence. Cells were paraformaldehyde fixed prior to blocking in 2% serum for 2 hours at 25°C. The primary antibody was diluted 1/100 and incubated with the sample for 18 hours at 4°C. Alexa fluor® 488 goat polyclonal to mouse Ig, diluted 1/1000 was used as the secondary antibody. In image, green is actinin staining and blue is DAPI is DNA staining.
IHC image of ab59468 staining in human breast carcinoma 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 ab59468, 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.