rev: December 2, 2004
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ANTIBODIES
(anti-Human and others as indicated)
RDI Division of Fitzgerald Industries Intl offers a wide line of antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.
cat#RDI-PRO10028 $438.00
Clone 5H9
Category Mouse monoclonal
Form/Purification purified IgG from culture supernatant
Ig Class IgG1
Immunogen Human uvomorulin
Clinical Application/Specificity Mab 5H9 specifically reacts with the 120 and 80 kDa ARC-1 protein in western blotting. In epithelia, E-cadherin is involved in the development and maintenance of cell layers during embryogenesis and in the adult tissue. Clustering of E-cadherin seems to occur via µ, ß and g catenins which link E-cadherin to cytoskeletal actin. Loss of E-cadherin expression seems to be correlated with the transformation state of carcinoma cells and with their metastatic potential.
Antigen Recognized in Species (tested so far) Human
Positive Control Cell line MCF-7, human small intestine
Application · Immunofluorescence microscopy
· Immunohistochemistry
· Immunoblotting
· Suitable for frozen tissue, for paraffin sections after microwave treatment
· NO proteolytic treatment
Dilution Buffer PBS with 1% BSA and 0.1% Na-Azide
Working Dilution 1:10 - 1:40
Storage At 2-8°C (undiluted) or in aliquots at -20°C
Volume 1 ml
References
Moll R et al: E-Cadherin expression in infiltrating ductal and lobular breast carcinoma (1993). Am J Pathol 143:1731-1742
INDIREKT IMMUNOPEROXIDASE STAINING ON FORMALIN-FIXED PARAFFIN- EMBEDDED TISSUE
- MICROWAVE TREATMENT -
1. Fix paraffin sections onto silanated oder polylysin-coated slides.
2. Dry overnight at 58°C.
3. Deparaffinize: dewax in xylol for 3 x 3 min; rehydrate in decreasing grades of ethanol: absolute, 96%, 70%, 50%, and dest. water for 3 min ea.
4. Microwave treatment incubate in plastic cuvette containing cold 10 mM Na-citrate buffer (pH 6.0) 5 x 5 min at 600
Watt in a microwave oven; let cool down after complete treatment to room temperature (15 min).
5. From this step onward it is essential that the sections do not dry out.
6. Rinse in dest. water and 2 x 5 min in PBS.
7. Optional: Block endogenous peroxidase with 0.6% H2O2/40% methanol-PBS for 30 min.
8. Rinse in PBS for 2 x 5 min.
9. Cover sections with 10 % heat-inactivated horse serum in PBS for 1 h (or alternatively with 5 % normal serum of the same species as the secondary antibody); depending on background staining, this step can be omitted or the serum concentration can be increased. Decant excess serum after incubation.1)
10. Incubation with optimal dilution of primary antibody (optimal dilution should be tested individually in each laboratory; start with a dilution of 1:10).
11. Rinse in PBS for 3 x 5 min.
12. Detection system: ABC method (Vector Laboratories); follow kit instructions; Counterstain with methyl green or hematoxylin
13. Dehydrate in increasing grades of ethanol, clear with xylol and mount with mounting medium
INDIRECT IMMUNOPEROXIDASE STAINING ON FROZEN SECTIONS
1. Fix sections on clean glass slides
2. Fixation in cold acetone or acetone/methanol for 5-10 min.
3. Let dry at RT
4. Rinse for 2 x 5 min in PBS
5. Optional: Block endogenous peroxidase with 0.6% H2O2/40% methanol-PBS for 30 min.
6. Rinse in PBS for 2 x 5 min.
7. Follow steps 10 -14 as given above
1) To reduce background, 2% skim milk powder may be added to the horse serum and all following incubations.
34 Junction Square Drive
Concord MA 01742-3049
USA
phone (800) 370-2222
or (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com
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