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The page below is a sample from the LabCE course Immunohistochemistry (IHC) Basics in Histology. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Buffers and Rinses

Wash buffers are used to rinse reagents from the tissue sections before adding new reagents. Wash buffers may also be used to mix secondary antibodies, link reagents, and labels in the staining process. It is important that the wash buffer is compatible and does a thorough job. They may also contain detergents that aid in the sheeting action for stringent washes. It is important when preparing buffers that:
  • The pH is optimal
  • It is pure buffer and not a mixture of buffers
  • All of the ingredients are fresh and not outdated
  • Proper storage is adhered to
Storage of buffers is as important to preserve stability and consistency when using them. Commonly, buffers are stored at 20° C, but there are a few that it is suggested to store at 25 - 27° C. Some concentrated buffer solutions that are diluted and mixed just before use may form crystals if stored in the refrigerator. These should be brought to room temperature while stirring to dissolve the salts before mixing the components together and checking the pH.