Immunohistochemistry FAQ Analysis

Problem	Cause	Recommendation
Background is too high	The quality of the primary antibody is not high	Use highly specific and high-titer primary antibodies
Background is too high	The concentration of the primary/secondary antibody is too high	Reduce primary/secondary antibody concentrations
Background is too high	The sealing is not complete	Increase the duration of protein blocking
Background is too high	The washing is not sufficient	Increase the number and duration of washes
Background is too high	The background can be reduced by adjusting the parameters of the fluorescence microscope.
Weak or no signal	No target protein or very low expression	Use cells or tissues with high expression levels
Weak or no signal	Poor cell permeability	Increase the duration or concentration of the permeabilization agent
Weak or no signal	Primary/secondary antibody concentration too low	Increase its concentration
Weak or no signal	Wrong secondary antibody selection (species mismatch)	Use a secondary antibody that is matched to the species of the primary antibody
Fluorescent light that goes out quickly	Fluorescein itself has poor light stability due to its intrinsic properties	Use a fluorescent secondary antibody with good light stability
Fluorescent light that goes out quickly	Seal with a common sealing agent	Seal the film with a fluorescent quencher
Cells have spontaneous fluorescence	Glutaraldehyde is used for fixation	Fluorescence quenching is performed after fixation and before fluorescent staining, such as with NaIO4, NaBH4, glycine, etc., and the level of autofluorescence is checked before staining
Cells have spontaneous fluorescence	The material itself (e.g., paraffin) has autofluorescence	Set up a negative control to eliminate background staining by reducing the parameters of the fluorescence microscope
Cells have spontaneous fluorescence	The content of molecules (e.g., riboflavin, cytochrome) in the cell components that can produce autofluorescence is high; the ratio of dead cells/live cells in the cultured cells is high

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