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Home > Method Guides > IHC Guides > Pitfalls and Troubleshooting

Pitfalls and Troubleshooting

Despite careful optimization, immunohistochemistry can present challenges. The following are common pitfalls and strategies for overcoming them:

Weak or No Staining

tips-and-hints-for-storage

Possible Causes	Solutions
Primary antibody needs antigen retrieval (AR) and no AR was done	Check information for AR on the data sheet of the primary antibody
Insufficient retrieval time, temperature or concentration	Increase retrieval time gradually in small increments, check buffer amount and temperature especially when using a microwave
Unsuitable antigen retrieval (AR) buffer	Test alternative AR buffers
Antibody concentration too low or antibody incubation time too short	Titrate primary and secondary antibody; extend incubation period
Incorrect secondary antibody or secondary system too weak	Check the secondary antibody; use ABC- or polymer-based secondary systems
Excessively long formalin/formaldehyde fixation of the tissue	See Tissue Fixation and possible Pitfalls
Incorrect long-term storage of tissue (IHC or IHC-Fr)	Store fixed tissue sections (IHC) at -20°C or fresh frozen tissue (IHC-Fr) at -80°C
Incorrect reconstitution or storage of antibodies	Read and follow the instructions on the data sheet of the antibodies. See Storage of antibodies See Reconstitution of antibodies

High Background Staining

Possible Causes	Solutions
Overexposure to heat or enzymes	Reduce retrieval time or enzyme concentration
Endogenous peroxidase or phosphatase activity	Include appropriate blocking steps (H₂O₂ for peroxidase, Levamisole for phosphatase)
Non-specific antibody binding	Titrate primary and secondary antibody
Insufficient or wrong blocking	Use normal serum from the host species of the secondary antibody or a serum-free block
Excessively long incubation with the substrate chromogenic solution	Check the staining reaction under the microscope
The ABC (avidin-biotin complex) system in immunohistochemistry reacts with endogenous biotin in tissues	Block endogenous biotin using an Avidin/Biotin blocking system or use a polymer-based secondary antibody
High autofluorescence of the tissue (e.g. lipofuscin, collagen or aldehyde/AR)	Perfuse the tissue before fixation (only animal tissues); reduce autofluorescence using a quenching kit; apply photobleaching; use spectral unmixing, try far-red dyes; if possible, use fluorescence lifetime imaging microscopy (FLIM), use the TSA method to increase Signal-Noise-Ratio.
Thick Tissue sections	Cut sections thinner

Tissue Detachment or Morphological Damage

Possible Causes	Solutions
Overheating or prolonged enzymatic digestion	Optimize retrieval conditions to prevent excessive damage
Insufficient adhesive coating on slides	Use adhesion slides (e.g. SuperfrostTMPlus)
Incompatible tissue processing techniques	Ensure proper tissue fixation and paraffin embedding, use a new blade during cutting and check settings of the cryostat or microtome
Excessive postfixation with methanol or acetone of fresh frozen tissue	Reduce postfixation time, pay attention to temperature (-20°C)

Non-Specific Binding and Artifacts

Possible Causes	Solutions
Ineffective blocking of endogenous biotin or IgGs	Use appropriate blocking agents (e.g., Avidin-Biotin for ABC methods)
Improper antibody titration	Optimize primary and secondary antibody dilutions
Buffer contamination	Ensure buffers and reagents are fresh and contamination-free
Poor tissue quality	Crush artifacts, folds, or necrotic areas in the tissue, as well as excessive thickness, can lead to false positive staining. Cut tissue with a sharp blade. Do not include physically damaged tissue areas in the evaluation
Tissue dried out during IHC	Keep the sample moist throughout the entire staining process; use a humid chamber for incubation steps
Not enough staining or buffer solution and too many tissue sections per staining well	Increase staining and buffer solution or reduce number of tissue sections per well
Non-homogeneous fixation or AR	Improve perfusion, fixation or AR time or concentration
Precipitations of primary or secondary antibody	Centrifuge antibody solutions before staining
Poor perfusion	Improve perfusion