Monoclonal Mouse Anti-Human Fascin Clone 55K-2

Code M3567

Intended use For Research Use Only. Not for use in diagnostic procedures.

Synonyms 55 kD -bundling , p55

Summary and explanation Human fascin is a highly conserved 55 kD actin-bundling protein which shares extensive sequence homology with fascin and the singed gene (sn) protein. Fascin, encoded by the human homolog for sn (hsn) gene, has been localized to microspikes and stress fibers of cultured cells where it is thought to be involved in the formation of bundles (1-3).

Reagent provided Anti-fascin is a mouse monoclonal antibody supplied in liquid form as tissue culture supernatant (containing fetal bovine serum) dialyzed against 0.05 mol/L Tris-HCl, pH 7.2, and 0.015 mol/L sodium azide.

Clone: 55K-2 Isotype: IgG1 Mouse lgG concentration mg/L: See label on vial.

Anti-fascin may be used at a dilution of 1:50 to 1:100 in the LSAB method determined on formalin-fixed, paraffin-embedded tissue. These are guidelines only; optimal dilutions should be determined by the individual laboratory.

Immunogen Fascin purified from HeLa cells (1,2).

Specificity Monoclonal anti-human fascin, clone 55K-2 (anti-fascin) reacts with a 55 kD protein in Western blots of HeLa, normal rat kidney (NRK) and gerbil fibroma cell lysates (2). Anti-fascin was also found to immunoblot bacterially expressed hsn gene product and a 55 kD protein from cell lysates of peripheral blood dendritic cells (3,4).

Materials required, but not supplied Refer to Dako’s General Instructions for Immunohistochemical Staining and/or the detection system instructions.

Precautions 1. The device is not intended for clinical use including diagnosis, prognosis, and monitoring of a disease state, and it must not be used in conjunction with patient records or treatment. 2. For professional users. 3. This product contains sodium azide (NaN3), a chemical highly toxic in pure form. At product concentrations, though not classified as hazardous, NaN3 may react with lead and copper plumbing to form highly explosive build-ups of metal azides. Upon disposal, flush with large volumes of water to prevent metal azide build-up in plumbing. 4. As with any product derived from biological sources, proper handling procedures should be used. 5. Wear appropriate Personal Protective Equipment to avoid contact with eyes and skin. 6. Unused solution should be disposed of according to local, State and Federal regulations.

Storage Store at 2–8 °C. Do not use after expiration date stamped on vial. If reagents are stored under any conditions other than those specified, the conditions must be verified by the user. There are no obvious signs to indicate instability of this product. Therefore, positive and negative controls should be run simultaneously with patient specimens. If unexpected staining is observed which cannot be explained by variations in laboratory procedures and a problem with the antibody is suspected, contact Dako Technical Support.

Specimen preparation Paraffin Sections Anti-fascin can be used on formalin-fixed, paraffin-embedded tissue sections.

The deparaffinized tissue sections must be treated with heat prior to the immunohistochemical staining procedure. For greater adherence of tissue sections to glass slides, the use of Silanized Slides (code S3003) is recommended.

When using the water bath method, preheat a Coplin jar containing 0.01 mol/L citrate buffer, pH 6.0, as well as a water bath to 95–99 °C. When the temperature has stabilized, place tissues into the Coplin jar containing the preheated buffer. Heat the tissue sections for 40 minutes. For improved staining results and a shorter incubation time, Target Retrieval Solution (code S1700) can be used in place of the 0.01 mol/L citrate buffer. Under these conditions the incubation time in the water bath may be reduced to 20 minutes. After thermal treatment, allow the jar with buffer and slides to cool for 20 minutes at room temperature. Rinse well with tap water and place slides into buffer.

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Cryostat Sections and Cell Smears Anti-fascin can also be used to label cryostat sections or cell smears.

Staining procedure Follow the recommended procedure for the detection system selected.

Staining interpretation The staining pattern for anti-fascin is cytoplasmic.

Tonsil: Positive cytoplasmic staining of follicular dendritic cells, and scattered parafollicular cells. Hodgkin's lymphoma: Positive cytoplasmic staining of Reed-Sternberg cells and vascular endothelia.

Performance characteristics Normal Cells Fascin has been localized by immunohistochemistry to the of a number of cell types. Fascin has been found to be strongly expressed in interdigitating reticulum cells (IDC) from the T-cell zones of the lymph node. Dendritic cells of the subcapsular area and of the reticular network also stain positively. Follicular dendritic cells (FDC) of the lymph node and spindle cells (interstitial dendritic cells) in perinodal tissue have been shown to exhibit variable immunostaining. Strong immunoreactivity has been observed in dendritic cells of the thymus, spleen and peripheral blood (veiled cells) (4,5). Fascin has also been demonstrated in the cytoplasm of histiocytes, smooth muscle cells, endothelial cells, squamous mucosal cells and lining cells of splenic sinuses (5,6).

Tumor Cells Fascin expression has been demonstrated in the cytoplasm of Reed-Sternberg cells and their variants. Anti-fascin was found to immunostain most Reed Sternberg cells and their variants in the majority of cases of Hodgkin’s lymphoma evaluated, including nodular sclerosis, mixed cellularity, lymphocyte depletion and unclassified types of Hodgkin’s disease. Among non-Hodgkin’s lymphomas and other lymphoid neoplasms, only a minority of cases were immunoreactive (5). In HIV-related lymphoid hyperplasia (HLP), anti-fascin was reported to stain both FDCs and IDCs in various subtypes of HLP. A decrease of fascin immunoreactivity in HLP was found to be associated with advanced disease and destruction of the dendritic network (7). Fascin immunostaining has also been demonstrated in a subset of mainly high grade breast carcinomas. Expression of fascin was found to correlate with tumor grade, Ki-67 proliferation index and p53 overexpression and to be inversely related to estrogen receptor expression (6).

References 1. Yamashiro-Matsumura S and Matsumura F. Purification and characterization of an F-actin-bundling 55-kilodalton protein from HeLa cells. J Biol Chem 1985; 260(8):5087 2. Yamashiro-Matsumura S and Matsumura F. Intracellular localization of the 55-kD actin-bundling protein in cultured cells: Spatial relationships with actin, alpha-actinin, tropomyosin, and fimbrin. J Cell Biol 1986; 103:631 3. Duh F-M, et al. cDNA cloning and expression of the human homolog of the sea urchin fascin and Drosophila singed genes which encodes an actin-bundling protein. DNA Cell Biol 1994; 13(8):821 4. Mosialos G, et al. Circulating human dendritic cells differentially express high levels of a 55-kd actin-bundling protein. Amer J Pathol 1996; 148(2):593 5. Pinkus GS, et al. Fascin, a sensitive new marker for Reed-Sternberg cells of Hodgkin’s disease: Evidence for a dendritic or B cell derivation? Amer J Pathol 1997; 150(2):543 6. Sun S, et al. Expression of fascin in breast correlates with tumor grade, increased cell proliferation and p53 tumor suppressor gene overexpression. US Can Acad Pathol 1997; 26A:133 7. Said JW, et al. The role of follicular and interdigitating dendritic cells in HIV-related lymphoid hyperplasia: Localization of fascin. Mod Pathol 1997; 10(5):421

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