Troubleshooting

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Background Staining

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

Weak Staining

Fixation Underfixed protein denaturation/degradation Overfixed cross-linking not reversed Incorrect fixative Insufficient fixative volume	Click here
Pretreatment Too short Incompatible reagent(s) and method	Click here
Primary Antibody Concentration too low Incubation too short Monoclonal generated	Click here
Detection Incubation too short Low sensitivity (LSAB) Improper reagent storage and handling	Click here
Chromogen Incubation too short Improper reagent storage and handling	Click here

No Staining

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

False Positive Staining

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

Tips

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

Tissue Preparation:

specimen handling
storage
fixation
processing
microtomy
slide preparation

Staining:

pretreatment
blocking
primary antibody
detection
chromogen
counterstain / coverslip

When performing a troubleshooting experiment, isolate one variable at a time.
Consult the manufacturer’s “Instructions-for-use” (IFU) to ensure you are following all product recommendations.
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