Application

Research Sub CategoryCytoskeleton

Immunocytochemistry: (5 µg/ml) Immunohistochemistry: (5 µg/ml) See IHC2011-6 for prediluted and detailed paraffin protocols.



Optimal working dilutions must be determined by end user.

Immunohysto/cyto chemistry Protocols

Ideal specimens are obtained from frozen sections from shock-frozen tissue samples. The frozen sections are dried in the air and then fixed with acetone at -20°C for 10 min. Excess acetone is allowed to evaporate at 15-25°C. Material fixed in alcohol and embedded in paraffin can also be used (2). Formaldehyde fixation will reduce or eliminate the intensity of staining depending on the conditions under which it is performed. Other fixation conditions must be first tested by the investigator.

It is advantageous to block unspecific binding sites by overlaying the sections with fetal calf serum for 20-30 min at 15-25°C. Excess of fetal calf serum is removed by decanting before application of the antibody solution.

Cytocentrifuge preparations of single cells or cell smears are also fixed in acetone. These preparations should, however not be dried in the air. Instead, the excess acetone is removed by briefly washing in phosphate-buffered saline (PBS).

Further treatment is then as follows:

Overlay the preparation with 10-20 µl antibody solution and incubate in a humid chamber at 37°C for 1 h.

Dip the slide briefly in PBS and then wash 3 x in PBS for 3 min (using a fresh PBS bath in each case).

Wipe the margins of the preparation dry and overlay the preparation with 10-20 µl of a solution of anti-mouse Ig-FITC or anti-mouse IgG-peroxidase solution and allow to incubate for 1 h at 37°C in a humid chamber.

Wash the slide as described above.

The preparation must not be allowed to dry out during any of the steps.

If using an indirect immunofluorescence technique, the preparation should be overlaid with a suitable embedding medium (e.g. Moviol, Hoechst) and examined under the fluorescence microscope. If a POD-conjugate has been used as the secondary antibody, the preparation should be overlaid with a substrate solution (see below) and incubated at 15-25°C until a clearly visible redbrown color develops. A negative control (e.g. only the secondary antibody) should remain unchanged in color during this incubation period. Subsequently, the substrate is washed off with PBS and the preparation is stained, if desired, with hemalum stain for about 1 min. The hemalum solution is washed off with PBS; the preparation is embedded and examined.

Substrate solutions:

Aminoethyl-carbazole: Dissolve 2 mg 3-amino-9-ethylcarbazole with 1.2 ml dimethylsulfoxide and add 28.8 ml 50 mM Tris-HCI, pH 7.3, and 20 µl 3% H 2 O 2 (w/v). Prepare solution freshly each day. Diaminobenzidine: Dissolve 25 mg 3,3′-diaminobenzidine with 50 ml 50 mM Tris-HCI, pH 7.3, and add 40 µl 3% H 2 O 2 (w/v). Prepare solution freshly each day.

Research CategoryCell Structure

Detect Desmin using this Anti-Desmin Antibody, clone DE-B-5 validated for use in WB, IH, IH(P), IH(P).

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Immunogen

Purified desmin.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Linkage

Replaces: 04-585

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Liquid in 0.02 M phosphate buffer, 0.25M NaCl with 0.1% sodium azide, pH 7.6.

Format: Purified

Quality

Western Blot Analysis: 1:1000 dilution

Specificity

The antibody reacts with desmin from human, pig, rat and taod. In tissue sections this antibody is used to stain skeletal, cardiac, visceral, and some vascular smooth muscle cells. Cell lines such as RD (ATCC CCL 136) and hamster BHK-21 are positive (Debus et al., 1983).

Storage and Stability

Maintain antibody refrigerated at 2-8°C in undiluted aliquots for up to 6 months. DO NOT FREEZE.