Analysis and handling of IHC common problems

Problem	Reason	Suggest
The result is too dark	The concentration of the primary antibody is too high or the incubation time is too long	Reduce primary antibody concentration or incubation time
The result is too dark	The incubation temperature is too high	Incubate at room temperature or 4 °C
The result is too dark	The incubation time of the HRP-labeled secondary antibody is too long	Reduce time
Non-specific staining in IHC results	The paraffin section is not completely dewaxed	Extend deparaffinization time
Non-specific staining in IHC results	Insufficient rinsing during the procedure	Increase the number and duration of washes
Non-specific staining in IHC results	The tissue is rich in endogenous biotin and peroxidase	Block with relevant reagent
Non-specific staining in IHC results	Antigenic ectopia occurs
Non-specific staining in IHC results	Insufficient protein blocking	Increase the protein blocking time
Weak color development in IHC results	The concentration of the primary antibody is too low or the incubation time is too short	Increase the concentration of the primary antibody or extend the incubation time
Weak color development in IHC results	The reagent has expired	Confirm the shelf life of the reagent before the experiment
Weak color development in IHC results	Excessive rinsing during the procedure	Rinse appropriately
No staining in IHC results	Expression of non-target antigens in the tissue
No staining in IHC results	mismatch between the species of the primary antibody and the secondary antibody	Verify the species origin of the primary antibody before the experiment
No staining in IHC results	mismatch between the chromogen and the HRP system	Verify the color development system before the experiment

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