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Immunohistochemistry-Paraffin - Protocol & Troubleshooting

Immunohistochemistry (IHC) protocols allow localization and measurement of specific proteins in tissue. Paraffin-embedded tissue sections sometimes allow for better preservation of tissue morphology. Paraffin sections also allow you to work with larger tissues and store them more easily. Here we will show you how to perform IHC using paraffin-embedded sections (IHC-P). It consists of a series of procedures including fixation, paraffin embedding, sectioning, and staining of the tissue.

We provide you with a simple protocol for IHC-P that you can refer to, which contains some troubleshooting tips and hints. These methods for paraffin section preparation and immunohistochemical staining should help put you on the right experimental track.

Solutions and Reagents

Stages Solutions and Reagents
Section Preparation Washing buffer, neutral buffered formalin, ethanol solution, xylene solution, liquid paraffin, antigen retrieval buffer
Antibody Staining Blocking buffer, primary antibody, labeled secondary antibody, dilution buffer, washing buffer, incubation buffer, chromogen
Mounting Mounting solution, washing buffer

Immunohistochemistry-Paraffin Procedure


1. Paraffin-Embedded Section Preparation

Prepare freshly dissected tissues and wash and cut them for use. Soak the tissue with a fixative for fixation. Neutral buffered formalin (NBF) is the usual fixative and the fixation time depends on the size of the tissue block and the type of tissue. Wash the tissues with buffer at the end of fixation. Dehydrate the tissues by soaking them several times with gradient concentrations of ethanol. Then wash the tissue several times using xylene. And immerse the tissue several times in liquid paraffin and keep it warm. Pour the paraffin wax into a mold, then transfer the tissue quickly into the mold and cool it at room temperature until the paraffin wax solidifies. Slice the paraffin-embedded tissues and incubate the sections in water to spread them. Mount the tissue sections on slides, dry them, and set them aside.

2. Dewaxing and Rehydration

Incubate the slides in xylene solution and repeat several times to ensure that the sections are completely dewaxed. Transfer the slides to ethanol solution and wash them several times at reduced concentrations. Then wash with washing buffer to remove the ethanol. Be sure not to allow the slide to dry out during this process.

3. Antigen Retrieval

Perform antigen retrieval to reveal antigenic epitopes. The most commonly used antigen retrieval is heat-induced epitope restoration (HIER). There are various methods of heating after the addition of antigen retrieval buffer, such as microwaving and boiling. You can read more about antigen retrieval to help determine the best conditions for your sample. When finished, cool the slide and wash it.

4. Antibody Staining

Select the ideal blocking reagent and incubate the sample with the blocking buffer. Incubation time and temperature are set on a case-by-case basis. When finished, wash the slide and remove the blocking buffer. For direct labeling, dilute the primary antibody appropriately and transfer the slide to the incubation buffer containing the primary antibody for incubation. Then wash the slides. For indirect assays, transfer the sections to the incubation buffer containing the secondary antibody and continue incubation. Wash the slide after completion to remove excess solution. Add the appropriate dilution of chromogenic agent to the slides and incubate away from light.

5. Counterstaining

The counterstaining step is optional. Counterstaining provides contrast with antibody staining to better distinguish the target signal. Select the ideal counterstains, incubate the sections, and rinse them upon completion.

6. Mounting and Detection

After staining, use the appropriate sealing solution to mount coverslips and seal the samples. Mounted slides can be stored permanently at room temperature. Observe the color of the antibody stain in the tissue section under the microscope.


IHC-P is a procedure for sectioning and staining tissue embedded in paraffin. Although it sounds easy, the reality is that there are many possible combinations of tissue types, antibodies, and assays, which can easily lead to various problems. If you need to perform some troubleshooting, please refer to our extensive troubleshooting guide. We solve your problem by adjusting a relatively small number of variables.

Tissue section problem

  1. Uneven tissue embedding problem. The uneven surface of the tissue section may result in the lost of important information. You should ensure that the blade is sharp enough and adjust the cutting speed to cut thicker sections.
  2. Tissue section detachment problem. Use positively charged or coated slides, or replace them with new slides. Be gentle when using antigen retrieval methods and avoid over-stirring the slide. During the fixation step, you can change the fixative or increase the fixation time.

No or inadequate staining

  1. Antigen causes. If the test slide is inadequately stained or unstained, while the positive control slide is adequately stained, the antigen may be the problem. The antigen is not present in the test tissue or is present but at a level below the limit of detection. Consider using an amplification procedure or increasing the primary antibody concentration, incubation time, or temperature. If antigen coverage is caused by over-fixation or under-fixation of tissue sections, a modified antigen retrieval protocol is required.
  2. Antibody causes. If the positive control is weak or unstained and the test slide is also weak or unstained, the antibody may be the cause. Check the antibody, including conditions such as dilution concentration, incubation time, and temperature. Pay attention to check if the secondary antibody and primary antibody are compatible because the test requires the use of a secondary antibody that will interact with the primary antibody.
  3. Reagent causes. Check and make sure that the buffer solution is not contaminated, and that the wash time is appropriate as a long-time wash can cause a reduced signal. It is necessary to check the relevant reagents such as buffers and chromogenic agents, and expiration dates, storage parameters, pH, and order of use are all included.
  4. Microscope causes. Check microscope parameter settings and increase the exposure time of the camera.

High background

  1. Section causes. Consider first if the tissue section is too thick and thinner sections can be prepared. Then check if sections are adequately fixed or if necrosis and autolysis are present. If any of these conditions are present, avoid sampling necrotic areas and ensure that the tissue is properly fixed. As well, inappropriate kinds of or the excess use of section adhesives can affect the background. Throughout the process, check and ensure that tissue sections are not dry.
  2. Dewaxing causes. You should repeat the dewaxing step several times to completely remove the paraffin from the sample.
  3. Antigen retrieval causes. You may have used an inappropriate antigen retrieval method. Please re-evaluate the antigen retrieval conditions and optimize the antigenicity of your sample.
  4. Blocking causes. Possible reasons for this are inadequate blocking of endogenous enzyme activity, biotin, or protein during the blocking phase. You can increase the concentration of the blocking agent or use a different blocking agent. In addition, you need to be aware that the blocking buffer should be made from a blocking serum of the same species.
  5. Primary antibody causes. Improper antibody concentration causes high background. You can re-titrate the primary antibody and select the appropriate concentration. Check the primary antibody incubation time, too long time can also cause this problem. You can shorten the incubation time. Clean sections and no antibody solution remaining should be ensured as well.
  6. Secondary antibody causes. As with the primary antibody, check the secondary antibody and label concentration and incubation time. It is important to note that the secondary antibody chosen needs to be unbound to tissue immunoglobulins. Clean sections and no antibody solution remaining should be ensured as well.
  7. Chromogen causes. If the chromogen concentration is too high and the reaction time is too long, you need to reduce the chromogen concentration and shorten the incubation time. It is also possible that the counterstain is masking the IHC reaction, and we suggest you replace a different counterstain. Clean sections and no excess stain remaining should be ensured as well.

We hope that this information about IHC-P above is useful to you as a reference guide. If you need more help with your experiments, please contact us for further assistance.


  1. Pal A. Immunohistochemistry. Protocols in Advanced Genomics and Allied Techniques, 2022: 95-117
  2. Ramos-Vara J A. Principles and Methods of Immunohistochemistry. Drug Safety Evaluation. Methods in Molecular Biology, 2017.

For research use only. Not intended for any clinical use.

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