Cytoskeletal preparation of HeLa cells (Debus et al., 1982).

Immunocytochemistry: 20 μg/mL

Immunohistochemistry: 20 μg/mL Not for formaldehyde fixed tissues.

Optimal working dilutions must be determined by end user.

Immunohistochemistry/Immunocytochemistry Protocols:

Ideal specimens are obtained from frozen sections from shockfrozen tissue samples. The frozen sections are dried in the air and then fixed with acetone at -15 to -25°C for 10 min. Excess acetone is allowed to evaporate at 15-25°C. Material fixed in alcohol and embedded in paraffin can, however, be used, see (4). For the immunohistochemical detection of cytokeratin No. 18 in tissue sections, the tissue should not be fixed in formaldehyde as this fixative markedly reduces the staining intensity of the cytokeratin filaments. lt 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 1h.

• Dip the slide briefly in PBS and then wash 3 times 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 an anti-mouse-IgG-FITC or anti-mouse-IgG-peroxidase antibody 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. lf using an indirect immunofluorescence technique, the preparation should be overlaid with a suitable embed-ding medium (e. g. Moviol, Hoechst) and examined under the fluorescence microscope.

lf 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.

Wash away the substrate with PBS and stain the preparation, if desired, with hemalum stain, for about 1 min. The hemalum solution is washed off with PBS, the peparation is embedded and examined.

Substrate solutions:

Aminoethyl-carbazole Dissolve 2mg 3-amino-9-ethylcarbazole with 1.2 mL dimethylsulfoxide and add 28.8 mL Tris-HCI, 0.05 M; 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 Tris-HCI, 0.05 M; pH 7.3, and add 40 μL H 2 O 2 , 3% (w/v). Prepare solution freshly each day.

This Anti-Cytokeratin 18 Antibody, clone CK2 is validated for use in IC, IH for the detection of Cytokeratin 18.

The antibody reacts with cytokeratin No. 18 from human. This antibody is used for staining research samples of a wide variety of simple epithelia and simple glandular epithelia (e. g. intestinal, respiratory and urinary epithelia) and transitional epithelia (e. g. bladder), but stratified squamous epithelia are not stained (e. g. esophagus, epidermis). Keratin filaments of cell lines such as HeLa cells are stained (Debus et al., 1982). For staining research samples of human tumor material with this antibody, see (Debus et al., 1984). For the cytokeratin pattern of different tissues, see (Moll et al., 1982).

Format: Purified

The lyophilized antibody is stable when stored at -20°C.Store the reconstituted antibody solution at 2-8°C, for up to 12 months.DO NOT FREEZE.

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

Replaces: 04-586

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