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Water for Immunohistochemistry

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Impact of Water

Water is ubiquitous in histology laboratories. It is used in water baths, floatation baths, and in most of the automated instruments present in the laboratory (stainers, tissue processors, etc.). Poor quality water may generate hard deposits or bacterial biofilms which may interfere with the proper functioning of these instruments. In addition, since water is the main component in many of the reagents prepared in the histology laboratory, water contaminants may interfere with the quality of the final slides.

In immunohistochemistry (IHC), relatively small volumes of water are used, especially if kits are used. Water is used for rinsing slides and glassware, to prepare buffers and reagent solutions, and in the heating devices used for the antigen retrieval (i.e. pressure cooker).

The following water contaminants may affect IHC results:
  • Alkaline phosphatase (bacterial)
    Calf intestine alkaline phosphatase is commonly used as a detection enzyme in IHC. If the water used in the IHC procedure contains bacteria, bacterial alkaline phosphatase may be released, and create interferences with the assay. Using an ultrafiltration cartridge (BioPak) at the point-of-use of the water purification system can ensure that the ultrapure water produced by the system does not contain any bacterial alkaline phosphatase.

  • Bacteria
    Bacteria and their by-products, in particular alkaline phosphatase, may generate artifacts on slides, and should be kept under control. They may also alter the quality of stock buffers and reagents, if they are stored.

  • Organics
    Organic molecules may interfere with the signal. In particular, polyaromatics and heterocyclics can interfere with fluorescent dyes, which are often used in IHC. In addition, organics can be used as a nutrient by bacteria, and lead to bacterial proliferation in buffer or reagent solutions, if they are stored.

  • Ions
    Ions, and in particular metals, can interfere with dyes and the antibodies used in IHC. Ionic concentrations and pH must be carefully controlled when working with antigen-antibody interactions, and the contribution of the water to these should be kept to a minimum.

  • Particles
    Particulate material may adhere to the tissue sections and generate artifacts on slides.


Ultrapure water (water with a resistivity of 18.2 MΩ•cm and a low TOC level), purified with a combination of purification technologies such as activated carbon, reverse osmosis, ion exchange, electrodeionization, ultraviolet photo-oxidation is suitable for this application. Using freshly purified ultrapure water, with an ultrafiltration cartridge (BioPak®) placed at the point-of-use of the purification system ensures extremely low levels of bacteria and bacterial alkaline phosphatase.

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