Immunohistochemistry Troubleshooting



1. General procedures
• Check the dilution of the primary antibody. For immunofluorescence or staining formalin-fixed, paraffin-embedded tissues, the concentration required may be higher than you expect.
• Try increasing the incubation time of the primary antibody, up to overnight at 4�°C if necessary.
• Check that the secondary reagents are working by testing with an alternativeprimary antibody that you know works in your system. 
2. Immunofluorescence
• Check that the fixation method used is correct for the antibody in use (see protocol or frequently asked questions section for more details of recommended fixation methods).
• If paraformaldehyde is being used as a fixative, check to see whether permeabilisation is required to allow the antibody to reach the target antigen.
• Check that the correct wavelength of light is being used to stimulate the fluorescent marker.
• Check that the samples have been protected from light throughout the staining procedure, as light will destroy the fluorochrome. 
3. Formalin-fixed, paraffin-embedded sections
• Confirm that the antibody being used can actually be used for this application (for more information, please see our 'frequently asked questions' section).
• Check to see whether an antigen-retrieval step is required. The use of formalin or paraformaldehyde can result in antigen masking, thereby preventing the antibody from recognising the target antigen. Treatment of the sections by heat or enzymatic digestion may be required to reveal the antigen. It is worth noting that antigen retrieval methods may or may not work to completely retrieve the antigen!
• If optimal staining is not obtained, try altering the method of antigen retrieval, as a different process may be better for your system.
• If possible, check to see that the antibody works for IHC of acetone-fixed, frozen tissue sections.
• Check to see if the antibody works in other techniques such as western blot. 
4. High Background
• Quench tissue with H2O2, block with serum from the same host species and dilute primary and secondary antibody in buffer containing control serum.
• Confirm that an appropriate blocking serum is being used.
• Pre-absorb the primary and secondary antibodies by adding 1-5% normal serum (from the same species as the sections being analysed) to the buffer being used to dilute the antibodies.
• Increase the blocking step to overnight at 4�°C
• Where appropriate, reduce the incubation times of the primary and secondary antibodies with the sections.
• Conduct appropriate controls e.g. secondary only control to determine which antibody is giving rise to the high background.
• Conduct a serial titration to determine the optimum dilutions required to give the best results.
• DAB precipitate on sections? Try a quick wash with de-ionised water after the PBS wash before you apply the DAB reagents.