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H Endogenous 142 Mouse IgG1

Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of unpermeabilized A-431 cells using DSG2 (10D2) Mouse mAb (IF Specific) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 148

Product Usage Information

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:100

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

DSG2 (10D2) Mouse mAb (IF Specific) recognizes endogenous levels of total DSG2 protein.

Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant protein specific to the amino terminus of human DSG2 protein.

Desmosomes are a class of intracellular junction that tightly link adjacent cells in mechanically stressed tissues such as the epithelium and myocardium (1). They derive their characteristic strength from the protein desmoplakin, which acts as a tether by binding the cytoplasmic component of the desmosome at its N terminus (2), while its C terminus is anchored to the intermediate-filament cytoskeleton (3). Desmogleins and desmocollins belong to the superfamily of cadherin proteins, the "glue" of the desmosome, as they are essential for strong cell-cell contacts (4). There are 4 types of desmogleins in humans, DSG1-4, and 3 types of desmocollins, DSC1-3. DSG2 is expressed in all desmosome bearing tissues, while other desmosome cadherin proteins have more specialized tissue expression. Research studies have shown that aberrant expression due to mutation of DSG2 is associated with arrhythmogenic right ventricular cardiomyopathy (5,6). Research has also shown that mutation and altered expression of DSG1 and 3 have been associated with autoimmune disorders such as Pemphigus, inherited disorders such as defective hair-follicle differentiation, and striate palmoplantar keratoderma, an epidermal-thickening disease (reviewed in 7).

1.  Stokes, D.L. (2007) Curr Opin Cell Biol 19, 565-71.

2.  Kowalczyk, A.P. et al. (1997) J Cell Biol 139, 773-84.

3.  Meng, J.J. et al. (1997) J Biol Chem 272, 21495-503.

4.  Nollet, F. et al. (2000) J Mol Biol 299, 551-72.

5.  Awad, M.M. et al. (2006) Am J Hum Genet 79, 136-42.

6.  Pilichou, K. et al. (2006) Circulation 113, 1171-9.

7.  Delva, E. et al. (2009) Cold Spring Harb Perspect Biol 1, a002543.

Entrez-Gene Id 1829
Swiss-Prot Acc. Q14126

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.

DSG2 (10D2) Mouse mAb (IF Specific)