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REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rabbit IgG
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Immunofluorescence (Frozen)

Confocal immunofluorescent analysis of the hypothalamic region of normal mouse brain (left) and a magnified image (right) using Vasopressin (D8T3K) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunofluorescence (Frozen)

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 9.5 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-Rabbit secondary antibodies:

    NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

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

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

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

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes 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 July 2016

Protocol Id: 151

Product Usage Information

Application Dilutions
Immunofluorescence (Frozen) 1:50

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

Vasopressin (D8T3K) Rabbit mAb recognizes endogenous levels of total Vasopressin protein.


Species Reactivity: Mouse
Species predicted to react based on 100% sequence homology: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro155 of human Vasopressin protein.

Vasopressin is a neuroendocrine peptide that is released to the circulation by magnocellular neurons whose cell bodies are mainly found in the paraventricular and the supraoptic nuclei of the hypothalamus. It was first isolated from pituitary gland extracts and synthesized in 1951 (1). Vasopressin acts by activating G protein-coupled, V1a, V1b (also known as V3) and V2 receptors and plays a fundamental role in the maintenance of water homeostasis. One of its main functions is body water retention (2), hence its alternative name antidiuretic hormone or ADH. Vasopressin also leads to increased arterial blood pressure by raising peripheral vascular resistance (3). Vasopressin is also involved in other physiological processes such as acute heart failure (4), pain (5), and metabolic syndrome (6).


1.  Turner, R.A. et al. (1951) J Biol Chem 191, 21-8.

2.  Robertson, G.L. (1976) Recent Prog Horm Res 33, 333-85.

3.  Aoyagi, T. et al. (2009) Kidney Int 76, 1035-9.

4.  Gilotra, N.A. and Russell, S.D. (2014) World J Cardiol 6, 1252-61.

5.  Luttinger, D. et al. (1984) Int Rev Neurobiol 25, 185-241.

6.  Saleem, U. et al. (2009) J Clin Endocrinol Metab 94, 2558-64.


Entrez-Gene Id 551
Swiss-Prot Acc. P01185


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.

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Vasopressin (D8T3K) Rabbit mAb