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Posted By on 12/13/2018

A Comprehensive Guide on Immunohistochemistry (IHC) Troubleshooting

A Comprehensive Guide on Immunohistochemistry (IHC) Troubleshooting

Let’s find out the comprehensive guide on IHC Troubleshooting Overstaining to enhance optimization. Let’s highlight the reasons when there is a need to check Troubleshooting:

High Background

  • Cleaning of sections was insufficient:  For this, wash at least 3 instances among steps.
  • Endogenous enzyme which includes peroxidase or alkaline phosphatase became active:  If so, then block endogenous enzyme activity by using 3% hydrogen peroxide  in methanol solution previous to incubation of primary antibody.
  • Washing of sections was inadequate:  If so, wash it at least 4-5 times between steps.
  • Mouse antibody was used on mouse tissues:  Treat tissue with Mouse on Mouse blocking reagent prior to the primary antibody incubation
  • Sections dried out:  Avoid sections being dried out.
  • Incubation temperature may be too high:  Incubate sections or cells at 4°C.
  • Overstaining due to over amplification:  Reduce amplification incubation time and dilute the secondary antibody.

 No Staining

  • Antibody turned into inactive due to flawed storage:  If so, then aliquot antibodies into tiny volumes and keep in the freezer as well and keep away from repeated freeze and thaw cycles.
  • Fixation of tissue changed into too long:  In this case, decline the duration of postfixation. In any circumstance, tissue has already been overfixed, optimized antigen retrieval way to unmask the epitope.
  • Deparaffinization was incorrect:  If Deparaffinize sections become longer, then use fresh xylene solution.
  • Antibody become unsuitable for IHC methods:  In a case, if it's been validated in IHC, and what form of IHC. Examine the antibody in a negative WB to make certain it isn't damaged.
  • Fixation of tissue was inadequate or improper: Increase duration of postfixation or try different fixatives.
  • Antibody concentration became insufficient:  Hike up the attention or execute a serial dilution check to decide the maximum dilution that offers the great sign to noise ratio. Or incubate longer at 4°C.
  • Eliminate system changed in incompatible:  Substitute with a distinctive substrate system that's well suited with enzyme.


  • Antibody concentration became too high:  If so, decline concentration or carry out a titration to determine the most useful dilution for antibody
  • Eliminate incubation time became too long:  Decline substrate incubation time
  • Incubation time turned into too long:  If so, decline incubation time
  • The incubation temperature turned into too excessive:  Decline incubation temperature
  • Sections dried out:  If so, make sure to avoid sections being dried out

To get more understanding about the IHC Embedding Optimization, open the official web portal of Boster Antibody and ELISA Experts. If you ever need support with your experiments, contact the Boster SupportTeam any time.

For more details visit our website: https://immunostaining.info/


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Pleasanton, CA 94566, USA


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