How can you troubleshoot immunohistochemistry problems?

2 Antibody Selection and Validation

The second step in troubleshooting IHC problems is to check the antibody selection and validation. The antibody is the key component of IHC that determines the specificity and sensitivity of the detection. You need to choose an antibody that is suitable for IHC and has been validated for your tissue type and species. You also need to use the appropriate concentration, diluent, and incubation time for your antibody to avoid over- or under-staining. You can optimize the antibody conditions by performing a titration experiment, where you test different dilutions of the primary and secondary antibodies and observe the staining quality. You can also use positive and negative controls, such as tissues with known expression or absence of the antigen, or blocking peptides, to verify the specificity of the antibody.

3 Blocking and Washing

The third step in troubleshooting IHC problems is to check the blocking and washing steps. Blocking is a process that reduces nonspecific binding of the antibodies to the tissue by adding a protein or a detergent to the buffer. Washing is a process that removes excess or unbound antibodies from the tissue by rinsing with a buffer. Both blocking and washing are important to minimize background staining and improve signal-to-noise ratio. You need to use a suitable blocking agent and buffer for your tissue and antibody, such as bovine serum albumin (BSA), serum, or Tween-20. You also need to wash the tissue thoroughly and frequently between each step, using a buffer that matches the pH and salt concentration of the antibody diluent.

4 Detection and Visualization

The fourth step in troubleshooting IHC problems is to check the detection and visualization methods. Detection is a process that amplifies the signal from the primary antibody by using a secondary antibody that is conjugated to an enzyme or a fluorophore. Visualization is a process that converts the signal into a colorimetric or fluorescent product that can be observed under a microscope. You need to choose a detection and visualization method that is compatible with your tissue and antibody, and that provides sufficient contrast and resolution. For example, you can use chromogenic detection with diaminobenzidine (DAB) or 3,3'-diaminobenzidine tetrahydrochloride (DAB) for brown staining, or alkaline phosphatase (AP) for red staining. You can also use fluorescent detection with fluorophores that match the excitation and emission wavelengths of your microscope. You can optimize the detection and visualization conditions by adjusting the concentration, incubation time, and substrate of the secondary antibody, and by using appropriate counterstains and mounting media.

5 Storage and Handling

The fifth step in troubleshooting IHC problems is to check the storage and handling of the tissue and antibody samples. Storage and handling are factors that can affect the stability and activity of the antigens and antibodies, and thus influence the staining results. You need to store and handle the tissue and antibody samples properly to prevent degradation, contamination, or evaporation. For example, you can store the tissue samples at -80°C or in a fixative solution, and the antibody samples at 4°C or -20°C, depending on the manufacturer's instructions. You also need to handle the tissue and antibody samples gently and carefully, avoiding repeated freeze-thaw cycles, exposure to light or heat, or excessive shaking or pipetting.

6 Documentation and Evaluation

The sixth step in troubleshooting IHC problems is to document and evaluate the staining results. Documentation and evaluation are essential to record the protocol, conditions, and outcomes of your IHC experiments, and to analyze the data and draw conclusions. You need to document and evaluate the staining results systematically and objectively, using standardized tools and criteria. For example, you can use a digital camera or a scanner to capture the images of the stained tissue sections, and a software or a scoring system to quantify the staining intensity and distribution. You can also use a checklist or a flowchart to identify the potential sources of error and variability, and to compare the results with the expected or published findings.

7 Here’s what else to consider

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