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"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.\r\n\r\nProblem: Lack of Staining\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tTest or Action\r\n\t\t\r\n\t\t\r\n\t\t\tLack of antigen.\r\n\t\t\tCheck protein expression by in situ hybridization (in some rare cases translation may be blocked even though mRNA is detected).\r\n\t\t\r\n\t\t\r\n\t\t\tAntibodies do not work due to improper storage.\r\n\t\t\tFollow 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 \u00B0C) and avoid repeated freeze-thaw cycles.\r\n\t\t\r\n\t\t\r\n\t\t\tInactive primary or secondary antibodies.\r\n\t\t\tTest reporter system independently to assess reagent viability.\r\n\t\t\r\n\t\t\r\n\t\t\tInadequate tissue fixation.\r\n\t\t\tTry increasing the fixation time or try a different fixative.\r\n\t\t\r\n\t\t\r\n\t\t\tTissue overfixation.\r\n\t\t\tReduce 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.\r\n\t\t\r\n\t\t\r\n\t\t\tIncompatible secondary and primary antibodies.\r\n\t\t\tUse 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.\r\n\t\t\r\n\t\t\r\n\t\t\tAntigen was destroyed before incubation with the primary antibody.\r\n\t\t\tIf quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody.\r\n\t\t\r\n\t\t\r\n\t\t\tEpitope altered during fixation or embedding procedure.\r\n\t\t\tTry restoring immunoreactivity through various antigen retrieval techniques.\r\n\t\t\tEmbed tissue at 58 \u00B0C or below.\r\n\t\t\r\n\t\t\r\n\t\t\tAntigen retrieval was ineffective.\r\n\t\t\tIncrease the time of treatment or change the treatment solution.\r\n\t\t\r\n\t\t\r\n\t\t\tReagents omitted or used in wrong order.\r\n\t\t\tRepeat staining and confirm that correct reagents are used and are added in the correct order.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\nProblem: High Background\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tTest or Action\r\n\t\t\r\n\t\t\r\n\t\t\tHigh concentration of primary and or secondary antibodies.\r\n\t\t\tTiter antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies.\r\n\t\t\r\n\t\t\r\n\t\t\tHydrophobic interactions of the antibody and proteins in the tissue.\r\n\t\t\tLower the ionic strength of the antibody diluent (particularly monoclonal antibodies respond well to reducing the salt concentration).\r\n\t\t\r\n\t\t\r\n\t\t\tNon-speci?c binding of primary and/or secondary reagents to tissues.\r\n\t\t\tUse 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.\r\n\t\t\r\n\t\t\r\n\t\t\tNon-specific binding of secondary antibody.\r\n\t\t\tUse an antibody that has cross-reactive IgG species removed (absorbed against sample species).\r\n\t\t\r\n\t\t\r\n\t\t\tTissue dried out.\r\n\t\t\tAvoid letting the tissue dry during the staining procedure.\r\n\t\t\r\n\t\t\r\n\t\t\tReagents sticking to old or poorly prepared slides.\r\n\t\t\tStart over with freshly prepared or purchased slides.\r\n\t\t\r\n\t\t\r\n\t\t\tBackground due to ionic interactions.\r\n\t\t\tIncrease the ionic strength of the diluent buffer.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\nProblem: Cell/Tissue Morphology is Destroyed\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tTest or Action\r\n\t\t\r\n\t\t\r\n\t\t\tAntigen retrieval methods are too harsh.\r\n\t\t\tEmpirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen.\r\n\t\t\r\n\t\t\r\n\t\t\tTissue sections falling off slide. (more common with frozen sections)\r\n\t\t\tIncrease fixation time. Empirically determine an additional or alternative fixative.\r\n\t\t\tUse freshly prepared, adequately charged slides.\r\n\t\t\r\n\t\t\r\n\t\t\tTissue section appears torn or folded. Air bubbles under section.\r\n\t\t\tRe-cut sections using a sharp blade, or ignore damaged areas when analyzing the results.\r\n\t\t\r\n\t\t\r\n\t\t\tPoor resolution of tissue morphology\r\n\t\t\tCut thinner tissue sections. Ice crystals may have destroyed morphology of frozen sections.\r\n\t\t\r\n\t\t\r\n\t\t\tUnderfixation has physically damaged the tissue or cells during the staining process\r\n\t\t\tIncrease fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio.\r\n\t\t\tCut smaller pieces of tissue for more thorough immersion fixation.\r\n\t\t\r\n\t\t\r\n\t\t\tAutolysis of tissue leading to staining of necrotic debris.\r\n\t\t\tIncrease the fixation time, ratio. Consider using cross-linking fixative.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\nProblem: Staining is Inappropriate\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tTest or Action \r\n\t\t\r\n\t\t\r\n\t\t\tFixation method is inappropriate for the antigen.\r\n\t\t\tTry a different fixative or increase the fixation time.\r\n\t\t\r\n\t\t\r\n\t\t\tAntigen retrieval may be inappropriate for this antigen or tissue.\r\n\t\t\tTry different antigen retrieval conditions.\r\n\t\t\r\n\t\t\r\n\t\t\tElectrostatic charge of the antigen has been altered.\r\n\t\t\tTry adjusting the pH or cation concentration of the antibody diluent.\r\n\t\t\r\n\t\t\r\n\t\t\tDelay in fixation caused diffusion of the antigen.\r\n\t\t\tFix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative.\r\n\t\t\r\n\t\r\n\r\n\r\n"^^<http://www.w3.org/2001/XMLSchema#string>
<https://www.rndsystems.com/resources/technical/troubleshooting-guide-immunohistochemistry>