IHC Troubleshooting: Weak Staining

Good results in IHC experiments depend on strong, specific staining of the target antigens. A good result can only be achieved when a sufficient quantity of primary antibody penetrates the sample and binds its target with high specificity, and enough secondary antibody with active enzymatic or fluorescent conjugate binds the primary antibody. When an IHC experiment results in faint or weak signal, it often has to be repeated, costing valuable time, money, and resources. Use this troubleshooting guide to identify and resolve the source of your weak signal:

Problem Cause Solution
Low enzyme activity Enzyme/substrate reaction impeded Deionized water can sometimes contain peroxidase inhibitors. Use Boster antibody diluent buffer to ensure enzymatic activity
Optimize substrate pH to increase reaction intensity
Buffer incompatible with enzyme Do not use phosphate buffer with AP system
Do not use sodium azide with HRP system
Insufficient anitbody activity Loss of antibody potency due to time or improper storage Run a positive control to verify antibody binding activity
Store antibodies according to manufacturer instructions
Inadequate antibody penetration Use a permeabilizing agent in the blocking and antibody diluent buffers
Antibody concentration too low Use a higher antibody concentration or incubate for longer
Antibody cannot detect target protein in native conformation Check the datasheet to make sure your antibody has been validated for IHC
Check that the antibody is applicable to your IHC sample (paraffin vs. frozen sections)
Perform western blot on both native and denatured forms to verify antibody binding
Problems with tissue section Insufficient deparaffinization Increase deparaffinization time
Use fresh dimethylbenzene
Inadequate antigen retrieval Reduce the length of fixation step
Use a different antigen retrieval method
Perform antigen retrieval for longer
Tissue section has dried out Make sure the tissue is covered in liquid at all times
Loss of signal over time Prepare slides using fresh tissue sections
Store slides at 4C
Do not bake slides