Technically, IHC and ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be identified and optimized for each individual IHC/ICC study. Optimization of IHC/ICC may also require troubleshooting a variety of factors. The tables below highlight common IHC/ICC issues and provide appropriate experimental actions. For further assistance, please contact our technical service department.
Problem: Lack of Staining
| Possible Source | Suggestion |
| Lack of antigen. | Check protein expression by in situ hybridization (in some rare cases translation may be blocked even though mRNA is detected). |
| Antibodies do not work due to improper storage. | Follow storage instructions on the datasheet. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70 °C) and avoid repeated freeze-thaw cycles. |
| Inactive primary or secondary antibodies. | Test reporter system independently to assess reagent viability. |
| Inadequate tissue fixation. | Try increasing the fixation time or try a different fixative. |
| Tissue overfixation. | Reduce the duration of the immersion or post-fixation steps. If immersion fixation cannot be avoided (for example, collection of postmortem tissues or biopsies in pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents. |
| Incompatible secondary and primary antibodies. | Use a secondary antibody that will interact with the primary antibody. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody. |
| Antigen was destroyed before incubation with the primary antibody. | If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody. |
| Epitope altered during fixation or embedding procedure. | Try restoring immunoreactivity through various antigen retrieval techniques. Embed tissue at 58 °C or below. |
| Antigen retrieval was ineffective. | Increase the time of treatment or change the treatment solution. |
| Reagents omitted or used in wrong order. | Repeat staining and confirm that correct reagents are used and are added in the correct order. |
Problem: High Background
| Possible Source | Suggestion |
| High concentration of primary and or secondary antibodies. | Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies. |
| Hydrophobic interactions of the antibody and proteins in the tissue. | Lower the ionic strength of the antibody diluent (particularly monoclonal antibodies respond well to reducing the salt concentration). |
| Non-speci?c binding of primary and/or secondary reagents to tissues. | Use blocking step just prior to primary antibody incubation (we use 1% bovine serum albumin with 10% normal donkey serum). Non-fat dry milk is another option. |
| Non-specific binding of secondary antibody. | Use an antibody that has cross-reactive IgG species removed (absorbed against sample species). |
| Tissue dried out. | Avoid letting the tissue dry during the staining procedure. |
| Reagents sticking to old or poorly prepared slides. | Start over with freshly prepared or purchased slides. |
| Background due to ionic interactions. | Increase the ionic strength of the diluent buffer. |
Problem: Cell/Tissue Morphology is Destroyed
| Possible Source | Suggestion |
| Antigen retrieval methods are too harsh. | Empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen. |
| Tissue sections falling off slide. (more common with frozen sections) | Increase fixation time. Empirically determine an additional or alternative fixative. Use freshly prepared, adequately charged slides. |
| Tissue section appears torn or folded. Air bubbles under section. | Re-cut sections using a sharp blade, or ignore damaged areas when analyzing the results. |
| Poor resolution of tissue morphology | Cut thinner tissue sections. Ice crystals may have destroyed morphology of frozen sections. |
| Underfixation has physically damaged the tissue or cells during the staining process | Increase fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio. Cut smaller pieces of tissue for more thorough immersion fixation. |
| Autolysis of tissue leading to staining of necrotic debris. | Increase the fixation time, ratio. Consider using cross-linking fixative. |
Problem: Staining is Inappropriate
| Possible Source | Suggestion |
| Fixation method is inappropriate for the antigen. | Try a different fixative or increase the fixation time. |
| Antigen retrieval may be inappropriate for this antigen or tissue. | Try different antigen retrieval conditions. |
| Electrostatic charge of the antigen has been altered. | Try adjusting the pH or cation concentration of the antibody diluent. |
| Delay in fixation caused diffusion of the antigen. | Fix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative. |