Have a look at IHC Troubleshooting Tips .pdf
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Tips or guide requires to following to target the main cause of a barrier with your protocol and test out
outcomes. Or this, it is recommended to identify the problem with your IHC staining from the following
• High Background
• Non-specific Staining
• Weak or No Staining
1. IN CASE HIGH BACKGROUND: Make sure to consider the following IHC Troubleshooting Tips if you
are tackling high background.
• If Deparaffinization is not abundant: Make sure to indulge in fresh xylene and keep the Deparaffinize
• In a case Antibody Concentration is too up: Then Dilute the primary and/or secondary antibody more.
• If blocking inadequate: Then hike up the blocking incubation period as well as considering to amending
the blocking agent.
• If inadequate washing: Then wash it properly. Make sure to follow the protocol guidelines while
indulging in washing steps.
2. IN A CASE OF NON-SPECIFIC STAINING: Make sure to consider the following tips if you are tackling
• If Antibody Concentrations are too up: Make sure to decline the concentration as well as the
3. IN CASE OF WEAK OR NO STAINING: Make sure to consider the following tips if you are tackling weak
or no staining.
• In case of not sufficient primary antibody: Make sure to use a higher concentration of antibody
• If primary and secondary antibodies are opposed: In this case, the secondary antibody should be
upgraded against the host of the primary antibody. Moreover, Isotypes must be compatible as well.
• If Deparaffinization is not enough: In this circumstance, make sure to use fresh xylene as well
Deparaffinize sections should be longer.
• If the tissue is dried out: Then samples should be kept covered in liquid during the staining process.
• If cells were not permeabilized: Then, use Methanol and acetone fixation to permeabilize cells
• If tissues are over constant: Then, decline the duration of fixation as well as indulge in using the
various antigen retrieval methods to unmask the epitope.
• If Incubation time is too small: Then hike up the duration of incubation of the primary antibody with
• In the case of Slide storage issues: Then samples must be imaged quickly after processing as the signal
declines over time.
• If Antibody Storage issues: Then no need to store Antibody as recommended. For this, there will be a
need for the new vial to be used.
• If protein is not available in the tissues being tested: In this, execute a positive control. Moreover, in a
circumstance, if the protein is available but not sufficient, then use an amplification process to hike up
Booster Antibody and Elisa experts are a prominent destination to get more guidance about the IHC
Troubleshooting problems. It has veteran experts that can guide you thoroughly about the IHC
Troubleshooting Tips until you become satisfied. Paraffin Embedding involves the steps fixation,
dehydration, transparent zing, immersion and embedding. Paraffin Embedded Tissue is sliced by a rotary
microtome to give a thickness of 2-7 µm.
To contact Booster Antibody and Elisa experts visit our website: https://immunostaining.info/