Immunohistochemistry is a complex experiment with multiple parameters that need to be considered and optimized. Our guide to IHC troubleshooting details the possible sources of common problems and how to combat them.
HRP, biotin and micro-polymer conjugated secondary antibodies
The primary antibody and the secondary antibody are not compatible.
Use a secondary antibody that was raised against the species in which the primary was raised (e.g., if primary is raised in rabbit, use an anti-rabbit secondary). Check that the isotype of the primary is recognized by the secondary.
Not enough primary antibody is bound to the protein of interest.
Use a higher concentration of antibody.
Incubate longer (e.g. overnight) at 4°C.
The antibody may not be suitable for IHC procedures as it may not recognize the native (3D) form of the protein.
Check the antibody datasheet to see if it has been validated in IHC, and what type of IHC (formalin/PFA fixation, fresh frozen etc.). Successful use of the antibody in immunocytochemistry (ICC) or immunoprecipitation (IP) is also a good indication that the antibody recognizes the native form of the protein.
Test the antibody in a native (non-denatured) WB to make sure it is still functional.
The primary antibody/secondary antibody/amplification kit may have lost its activity due to improper storage, improper dilution or multiple cycles of freeze/thaw.
The protein is not present in the tissue of interest.
Run a positive control recommended by the supplier of the antibody.
The protein of interest is not abundant in the tissue.
Use an amplification step to maximize the signal. Find out more about indirect detection and amplification here.
The secondary antibody was not stored in the dark (if your detection system is fluorescence microscopy).
Always protect the secondary antibody from exposure to light.
Deparaffinization may be insufficient.
Deparaffinize sections longer and use fresh xylene.
Fixation procedures (using formalin and paraformaldehyde fixatives) may be modifying the epitope the antibody recognizes.
Use different antigen retrieval methods to unmask the epitope (heat mediated with pH 6 or pH 9 buffer, enzymatic, etc.).
Fix the sections for a shorter time.
The antibody cannot penetrate the nucleus, where the protein is located (nuclear protein).
Add a strong permeabilizing agent like Triton™ X-100 to the blocking buffer and antibody dilution buffer. See our protocol about permeabilization techniques.
The PBS buffer is contaminated with bacteria that damage the phosphate groups on the protein of interest (phosphorylated proteins).
Add 0.01% azide to the PBS antibody storage buffer or use fresh sterile PBS.
Blocking of non-specific binding might be absent or insufficient.
Increase the incubation period with the blocking agent and consider changing blocking agent. If using serum, Abcam recommends 10% normal serum of the species of the secondary antibody for 1 h. Alternatively, try a commercial blocking buffer. Another option is to try a secondary antibody that has been pre-adsorbed against the immunoglobulin of the species of your samples.
The primary antibody concentration may be too high.
Titrate the antibody to the optimal concentration, dilute the antibody further and incubate at 4°C (a slow but targeted binding is best).
Incubation temperature may be too high.
Incubate sections at 4°C.
The secondary antibody may be binding non-specifically.
Run a secondary control without primary antibody.
If you see staining with your secondary only, change your secondary or use a secondary antibody that has been pre-adsorbed against the immunoglobulin of the species of your samples.
Tissue not washed enough, fixative still present.
Wash extensively in PBS between all steps.
Endogenous peroxidases are active.
Use enzyme inhibitors ie, levamisol (2mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
Fixation procedures (using formalin or paraformaldehyde fixatives) are causing autofluorescence (if detection system is fluorescence microscopy).
Formalin/PFA usually autofluoresce in the green spectrum, so try a fluorophore in the red range.
Use a fluorophore in the infrared range if you have an infrared detection system.
Too much amplification (indirect technique).
Reduce amplification incubation time and dilute the secondary antibody or amplification kit reagent.
Too much substrate was applied (enzymatic detection).
Dilute substrate more and reduce substrate incubation time.
The chromogen reacts with PBS present in the tissue sample (enzymatic detection).
Use Tris buffer to wash sections prior to incubating with the substrate, then wash sections/cells in Tris buffer.
Permeabilization has damaged the membrane and removed the membrane protein (membrane protein).
Use a less stringent detergent (eg Tween® 20 instead of Triton™ X-100).
Primary/secondary antibody concentration may be too high.
Try decreasing the antibody concentration and/or the incubation period. Compare signal intensity against tissue that does not express the target protein.
Endogenous peroxidases are active.
Use enzyme inhibitors ie, Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
The primary antibody is raised against the same species as the tissue stained (eg mouse primary antibody tested on mouse tissue). When the secondary antibody is applied, it binds to all the tissue as it is raised against that species.
Use a primary antibody raised against a different species than your tissue.
Use a commercially available kit with proprietary reagents (e.g., our mouse on mouse kit).
The sections/cells have dried out.
Keep sections/cells at high humidity and do not let them dry out.
Poorly resolved or damaged tissue morphology
Antigen retrieval methods may be too harsh.
Vary antigen retrieval procedure or try different antigen retrieval methods.
The tissue may have been underfixed.
Increase fixation time.
Increase ratio of fixative to tissue.
Cut smaller pieces of tissue for more efficient fixation (fixation by immersion).
Tissue sections are falling off the slide (frozen sections).
Increase fixation time.
Try an alternative fixative.
Use freshly prepared slides.
Tissue sections are torn or folded, or air bubbles are visible under the sections.
Re-cut sections using a sharp blade.
Study areas of tissue that are unaffected. Localize reagents using a PAP pen.
The tissue morphology is poorly resolved.
Cut thinner tissue sections.
Ice crystals may have destroyed morphology of sections - re-cut and freeze rapidly (frozen).