Solving IHC experimental difficult problems: common problems and coping strategies

Immunohistochemistry (IHC) is an experimental technique used to detect the expression of specific proteins in tissues or cells. This technology is very important in medical research and diagnosis, but some problems may be encountered during the experiment. Let Xiaoai take you to see the coping strategies of common problems ~

 

No staining

 

1. Primary and secondary antibodies are incompatible

-use of anti-primary antibody species-derived secondary antibodies (e.g., if the primary antibody is rabbit-derived, use anti-rabbit secondary antibodies). Confirm whether the secondary antibody recognizes the subtype of the primary antibody.

 

2. There is not enough primary antibody to bind to the target protein

-Use a higher concentration of antibody.

Prolonged incubation at-4 °C (e.g. overnight incubation).

 

3. Antibodies may not be suitable for IHC because they may not recognize the native (3D) form of the protein

-Check the antibody instructions for use to confirm if the antibody is IHC validated and the type of IHC validated (formalin or PFA fixed, frozen sections, etc.). Successful use of antibodies in ICC or IP can also indicate that the antibodies can recognize the protein in its native form.

-The antibody is tested in native (non-denatured) WB to ensure it is still effective.

 

4. The primary antibody/secondary antibody/signal amplification kit may lose its activity due to improper storage, improper dilution or repeated freezing and thawing

-Set up positive controls to ensure that these reagents are still effective.

 

5. The protein is not present in the target tissue

-Set up positive controls in the literature or recommended by the antibody supplier.

 

6. The target protein is not abundant in tissues

-Using a signal amplification step to maximize the signal.

 

7. The secondary antibody is not stored in the dark (when performing fluorescence detection)

-Always avoid exposure of secondary antibodies to light.

 

8. Dewaxing may be insufficient

-Extend the dewaxing time of the slices, using freshly formulated xylene.

 

9. The immobilization step may change the epitope recognized by the antibody

-Different antigen repair methods are used to expose antigen epitopes (including thermal antigen repair, enzymatic antigen repair, etc. using a buffer with a pH of 6 or 9).

-Shorten slice fixation time.

 

10. Antibodies cannot penetrate the nucleus (nucleoprotein) where the protein is located.

-Add a potent permeable agent (e.g. Triton) to the blocking buffer and antibody dilution ™ X-100, Art. No.: abs9149).

 

11. PBS buffer is contaminated by bacteria, which will destroy the phosphate group (phosphorylated protein) on the target protein.

-Add 0.01% azide to PBS antibody storage buffer or use freshly formulated sterile PBS.

 

12. Incomplete antigen repair. For formaldehyde-fixed tissues, sufficient antigen repair must be used to open the antigen epitope to facilitate binding to antibodies

-It is recommended to try microwave repair with high heat 4 times * 6min. Someone has done experiments, and this is the optimal time and number of times. If not, it can also be repaired under high pressure.

 

Non-specific staining

 

1. The concentration of primary antibody/secondary antibody may be too high

-Attempt to reduce the antibody concentration or shorten the incubation time. The signal intensity is compared to that of tissues that do not express the target protein.

 

2. Endogenous peroxidase is active

-Use of enzyme inhibitors, i.e. levamisole (2 mM) for AP, or H2O2 (0.3% v/v) for peroxidase.

 

3. Use a primary antibody derived from the same species as the stained tissue (for example, mouse primary antibody is used on mouse tissue). When the secondary antibody is used, since the secondary antibody is targeted to the tissue species, it binds to the immunoglobulin or Fc fragment of the protein in the tissue

-using primary antibodies of different species origin than the tissue section, or blocking with F (ab) fragment secondary antibodies.

 

4. Sections/cells have been dried

-Keep sections/cells highly moist and do not allow them to dry out.

 

5. Polyclonal antibodies for primary antibodies are prone to non-specific staining

-It is recommended to try monoclonal antibodies.

 

6. Non-specific components bind to antibodies

-This requires enhancing the blocking effect by extending the blocking time of secondary antibody-derived animal immune serum and appropriately increasing the concentration.

 

Unclear or damaged tissue morphology

 

1. Antigen repair methods may be too harsh

-changing the antigen repair step or trying a different antigen repair method.

 

2. The tissue may not be adequately fixed

-Prolonged fixation time.

-Increase the ratio of fixative to tissue.

-Cutting smaller pieces of tissue for more effective fixation (immersion fixation).

 

3. The tissue section falls off the slide (frozen section)

-Prolonged fixation time.

-Try replacing the fixative.

-Use freshly prepared glass slides.

 

4. The tissue section is torn or folded, or air bubbles can be seen under the section

-Re-slice using a sharp blade.

-Study unaffected tissue areas. Use a PAP pen (Cat.: abs929) to locate the reagent.

 

5. The tissue morphology is difficult to distinguish

-Cut the tissue sections thinner.

-Ice crystals may disrupt slice morphology

-Re-slice, quick freeze (frozen sections).

 

6. Tissue autolysis

-Prolonged fixation time.

-increasing the proportion of fixative relative to tissue.

-Attempt to use a crosslinked fixative.

 

High background

 

1. Failure to block non-specific binding or insufficient blocking

-Extend the blocking time and consider changing the blocking reagent. If serum is used, we recommend blocking with 10% normal serum of the secondary antibody species for 1h. Alternatively, commercially available blocking buffers are attempted, or secondary antibodies pre-adsorbed with immunoglobulin of the sample species are used.

 

2. The concentration of primary antibody may be too high

-The optimum concentration of antibody was tested, the antibody was diluted in a gradient and incubated at 4 °C.

 

3. The incubation temperature may be too high

-The sections were incubated at-4 °C.

 

4. The secondary antibody may have non-specific binding

-A secondary antibody control without primary antibody was used.

-If staining is observed when the secondary antibody is used alone, the secondary antibody should be replaced, or a secondary antibody pre-adsorbed with immunoglobulin of the sample species should be used.

 

5. Insufficient tissue washing; Fixative remains present

-Between steps, the tissue was thoroughly washed with PBS or TBS. A surfactant, such as 0.1% Triton, is added.

 

6. Endogenous peroxidase is active

-Use of enzyme inhibitors, i.e. levamisole (2 mM) for AP, or H2O2 (0.3% v/v) for peroxidase.

 

7. The immobilization step results in autofluorescence (if fluorescence detection is used)

-Formalin/PFA generally produces autofluorescence in the green spectrum, so it is possible to try to use fluorophores in the red spectral range.

-If an infrared detection system is available, a fluorescent group in the infrared range is used.

8. Autofluorescence in tissues (if fluorescence detection is used)

-If autofluorescence does not need to be preserved, a tissue autofluorescence quencher can be used (Cat.: abs9860)

 

9. Excessive signal amplification (indirect detection technology)

-Shorten the incubation time of the signal amplification reagent and dilute the secondary antibody or the signal amplification reagent.

 

10. Too much substrate (enzyme detection)

-further dilution of the substrate, or shortening the incubation time of the substrate.

 

11. Chromogen reacts with PBS present in tissue samples (enzyme detection)

-Sections are washed with Tris buffer first, then incubated with substrate, and then sections/cells are washed in Tris buffer (special note, applicable to AP only).

 

Although IHC experiments are technically demanding, high-quality experimental results can be obtained through careful experimental design, strict operation procedures, and timely problem solving. Faced with the problems in IHC experiments, researchers need to be patient and meticulous, and constantly optimize the experimental conditions to ensure the success of the experiments.

 

Recommended small hobbies:

Item number	Product Name	Specifications
abs957	Ready-to-use high efficiency immunohistochemical secondary antibody kit	5mL
abs996	Ready-to-use immunohistochemical secondary antibody kit (universal type for pika and rabbit)	5mL
abs997	Ready-to-use immunohistochemical secondary antibody kit (anti-mouse)	5mL
abs998	Ready-to-use immunohistochemical secondary antibody kit (anti-rabbit)	5mL
abs9792	Histochemical double staining kit (universal for pika and rabbit)	50T

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.net

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