Need To Know About Immunohistochemistry Blocking | Immunostaining

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Immunohistochemistry (IHC)
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Introduction

• Immunohistochemistry (IHC) combines histological,
  immunological and biochemical techniques for the
  identification of specific tissue components by
  means of a specific antigen/antibody reaction
  tagged with a visible label. IHC makes it
  possible to visualize the distribution and
  localization of specific cellular components
  within a cell or tissue.

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Inactivation and Blocking

• Inactivation
• When either the horseradish peroxidase (HRP) or
  alkaline-phosphatase (AP) system is applied for
  IHC, activation of endogenous enzymes should be
  blocked or inhibited to avoid producing
  non-specific binding.
• Endogenous HRP Inactivation
• Incubate the paraffin embedded section in 3
  H2O2 for 10 min
• Incubate the frozen section or cell section in
  solution composed of methanol and 3 H2O2 (v/v
  41) for 30 min.

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Continue . . . . . . . . . .

• Endogenous AP Inactivation
• Incubate the sample section in 0.1 mM Levamisole
• Note Levamisole cannot inhibit the AP
  activation of endogenous intestine tissue

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Blocking

• Residual sites on the tissue section may bind to
  secondary antibody and produce follow-up false
  positive results. Therefore, serum from the same
  species as the secondary antibody is commonly
  used for blocking.
• Animals autoantibody in the serum can bind to
  the sites in advance. Blocking should be done at
  room temperature for 10-30 min (avoid excessive
  blocking).

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