Background Staining

Fixation
Underfixed autolysis
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Pretreatment
Too short/long
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Blocking
Incubation too short (or none at all)
Wrong blocking reagent
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Primary Antibody
Concentration too high
Incubation too long
Polyclonal or ascites generated
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Detection
Incubation too long
High sensitivity (2-step polymers)
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Chromogen
Incubation too long
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Weak Staining

Fixation
Underfixed protein denaturation/degradation
Overfixed cross-linking not reversed
Incorrect fixative
Insufficient fixative volume
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Pretreatment
Too short
Incompatible reagent(s) and method
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Primary Antibody
Concentration too low
Incubation too short
Monoclonal generated
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Detection
Incubation too short
Low sensitivity (LSAB)
Improper reagent storage and handling
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Chromogen
Incubation too short
Improper reagent storage and handling
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No Staining

Fixation
Underfixed protein denaturation/degradation
Incorrect fixative
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Microtomy
Insufficient slide baking tissue loss
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Pretreatment
Too short
Incompatible reagent(s) and method
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Primary Antibody
Concentration too low
Incubation too short
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Detection
Improper reagent storage
Reagent contamination or mix-up
Incubation too short
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Chromogen
Improper reagent storage and handling oxidation
Reagent contamination
Incompatible enzyme-chromogen interaction
Incubation too short
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False Positive Staining

Fixation
Incorrect/non-recommended fixative
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Mictrotomy
Artifacts, folds, etc
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Pretreatment
Non-recommended reagent(s) or method
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Blocking
Incubation too short (or none at all)
Wrong blocking reagent
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Primary Antibody, Detection, Chromogen
Concentration too high
Incorrect optimization (titration)
Cross-contamination
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Tips

  1. Consider the entire staining process and understand the impact of all potential variables to include:

  2. Tissue Preparation:
    • specimen handling
    • storage
    • fixation
    • processing
    • microtomy
    • slide preparation
    Staining:
    • pretreatment
    • blocking
    • primary antibody
    • detection
    • chromogen
    • counterstain / coverslip

  3. When performing a troubleshooting experiment, isolate one variable at a time.
  4. Consult the manufacturer’s “Instructions-for-use” (IFU) to ensure you are following all product recommendations.
  5. Contact your trusted technical support representative sooner rather than later.
    (You can contact your representative or use the following form to submit your questions.)

Troubleshooting Questions

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