Five Tips for IHC Troubleshooting

IHC troubleshooting for endogenous peroxidase, antigen retrieval, serum blocking, antibody incubation, and DAB staining is specified below.

1. Inactivation Time and Concentration of Endogenous Peroxidase

Usually, 3% hydrogen peroxide takes shorter inactivation time and can be kept for 10min in the dark. 0.3% hydrogen peroxide can properly extend blocking time(10-30 min).

It's suggested to prepare hydrogen peroxide in methanol which is better than double distilled water or PBS. Methanol can protect antigen and fix tissues. Overlong incubation for hydrogen peroxide can easily result in dropping.

Store the prepared hydrogen peroxide at 4ºC in the dark.

2. Antigen Retrieval Condition

Antigen retrieval is necessary for paraffin-embedded slices. Most formaldehyde fixed tissues are required for antigen retrieval before staining. Methylene bridge produced during fixing process results in protein crosslinking and shields antigen sites.

Common methods include heat induced antigen retrieval (HIER) and protease-induced epitope retrieval(PIER). Two common buffers are recommended: 10mM citrate buffer(pH 6.0) and 0.5mM Tris-EDTA buffer(pH 9.0). Suitable buffers can follow manufacturer's instructions. Usually, Tris-EDTA buffer is applicable to most phosphotyrosine-specific antibodies. Citrate buffer applies to other antibodies.

Microwave antigen retrieval is frequently used in the lab.

3. Serum Blocking Protocol

Some spare areas in the slice can nonspecifically absorb antibodies, leading to the false-positive result. Blocking is required for the incubation of primary antibody.

Blocking with a serum homologous to the species of the secondary antibody is required. Animal's autoantibody in the serum can bind to sites with cross reaction in tissues in advance. Otherwise, the binding with secondary antibody will result in background.

Calf serum, BSA or goat serum can be also used but should be different from the host of primary antibody.

4. Comparison of Antibody Incubation Conditions

Incubation temperature: 4ºC, room temperature, 37ºC. The best effect is 4ºC overnight.

Incubation time: related to temperature and antibody concentration. Incubate at 37ºC for 1-2 h or 4ºC overnight.

5. IHC DAB Troubleshooting 

DAB staining time is irregular and controlled under the microscope. If a light brown staining appears, washing is allowed.

DAB staining time is very short(e.g. seconds or tens of seconds). The dark chocolate-brown appears, showing the higher antibody concentration or overlong incubation. Thus, antibody concentration or incubation time should be decreased.

If the dark background appears in a very short time, the previously blocked non-specific protein may be incomplete. Then, the blocking time should be extended.

Positive staining appears after a long DAB staining time(e.g. more than ten minutes), showing lower antibody concentration, shorter incubation(4ºC overnight is better), or overlong blocking.

6. Recommended IHC Antibodies

REFERENCES

[1]Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips, PMID: 27809448.
[2]Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories, PMID: 18599844.