How to Make Antibody Dilutions for IHC

Immunohistochemistry (IHC) is a specialist technique used by scientists to visualize cells within tissues that have been placed on a slide and stained with an appropriate antibody that recognizes a protein that is unique to the cell or tissue under investigation. Antibody dilutions (titrations), must be performed using the experimental conditions specified in the user's own protocol, not that of the antibody's manufacturer, since the two may not be identical.

Things You'll Need

• Cells to stain
• Antibody
• Dilution buffer ("diluent")
• Pipettes and tips
• Microfuge tube
• Aluminium foil
• Lab markers and tube labels
• Ice
• light-protected slide box

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Instructions

• 1

  Check and prepare the antibody. Verify that the antibody has been raised in the correct host, and reacts with the exact version of the protein (known as the isoform, or splice variant) that is being tested. Using a pipette, carefully take a 10 microliter portion of this and label as 'personal stock'.

  If only very small volumes of antibody are available, skip this step.

  Keeping a personal stock of antibody is routine practice in all laboratories as it prevents cross-contamination of the original stock. Dispense this into a light-protected microfuge tube, or a normal microfuge tube which is wrapped in aluminium foil to protect from direct light.

  Keep this tube on ice, preferably in an Esky with an air-tight lid, again to minimize light, which can destroy the antibody. Make a note of the original stock's concentration (e.g. 100 Units per milliliter, 1 milligram per microliter and so on) on the tube, including the name of the antibody and what, if any, buffer it is diluted in (e.g. serum, saline, or water etc).

• 2

  Prepare the dilution buffer, also known as the antibody diluent. Check to make sure this is compatible with the antibody and the immunohistochemistry procedure being used, for example, it should not obscure any fluorescence or inhibit any subsequent secondary antibody labeling. Make a standard immunohistochemistry dilution (also the blocking buffer) by mixing 1X phosphate buffered saline with 1% Triton-X100, 10% fetal calf serum and 0.2% sodium azide.

• 3

  Titrate the antibody. With the given concentration of antibody in the personal stock (expressed as concentration units such as microgram per microliter), determine the volume of antibody required to obtain 10 micrograms of antibody.

  Set up a dilution series by pipetting a volume equivalent to 2 microgram of antibody (e.g. X microliters), in 100 microliters of diluent and mix thoroughly by pipetting up and down. From this starting dilution, take the same X microliters and add that to a subsequent 100 microliters of diluent. Repeat this until 8-10 dilutions (or a series) is made up.

  The last tube will therefore have the lowest concentration and be the "saturating" concentration that will generate the highest level of signal during the experiment. However, the ideal concentration will be slightly more concentrated than this, so that not too much signal is produced, which can cause background errors.

  Label the tubes correctly with the new diluted concentrations.