Experimental Operation Guideline-Trouble Shooting of Immunohistochemistry (IHC)

Cloud-Clone Corp. Publish date: 2015-09-28

Immunohistochemistry (IHC) is also called as immunocytochemistry (ICC). It is a immunological method for qualitative and quantitative detection, as well as location of antigen in situ in cells of a tissue section, using specific and labeled antibody. It bases on the reaction of antigen and antibody, together with the color reaction of histochemistry. It skillfully combines the specificity of immunological reaction, the visibility of histochemistry and the sensitivity of molecular technology. For antigens (such as protein, peptide, enzyme, hormone, pathogen and receptor, etc) can be detected in cellular or subcellular level by visualization and amplification using microscope, it provides a powerful tool to diseases diagnose, identification and research on the mechanism of the diseases as it is updated to the dynamic observation of structure, function and metabolism from stationary morphologic description.

However, it is not easy to get a high quality staining IHC section. As there are a lot of procedures during the process of slicing and staining. Each step should be strictly controlled, otherwise final result will be affected. Common problems during the experiment are analyzed and solutions are provided in order to help researchers to improve the success rate.

Table 1.Trouble Shooting

Problem		Possible reason	Solutions
No staining		Invalid reagent Reagents omitted reagents used in wrong order	Repeat the experiment and set a positive control.
		The protein of interest is not present in the tissue or the concentration is too low	Search for related papers or database to confirm the protein of interest exists in the tissue. Set a positive control to verify the result.
		Incompatible secondary and primary antibodies.	Confirm the species of primary and secondary antibody. If the primary antibody is generated in rabbit, secondary antibody against IgG of rabbit should be chosen. Check the type of primary antibody. If it is a mouse IgG1, second antibody should be against mouse IgG1.
		Inadequate primary antibody is bound to the protein of interest	Increase the concentrate of antibody, incubate longer (e.g. overnight at 4°C)
		Antigen retrieval is not processed	Antigen retrieval is essential in certain tissue before detecting antigen.
		The epitope may be modified during fixing and embedding	Use several methods for antigen retrial. The temperature should be lower than 58 ° during embedding.
		Washing solution and reaction reagent are not compatible	Adjust the PH of solutions. Substrate solution should not contain sodium azide.
Weak staining		Antibody concentration may be too low	Increase the concentrate of antibody, the incubation time should not be less than 60 minutes.
		Incubation time is too short	No less than 60 minutes for incubation.
		The concentration of target protein in the specimen is too low	Search for related papers or database to confirm the protein of interest exists in the tissue. Set a positive control to verify the result.
		Too much washing solution is remained in the section during the operation	Remove excess buffer in the section before adding new reagent (Avoid drying out).
		The section is not kept horizontal during incubation, resulting in the lost of antibody	Keep the section horizontal during incubation.
		Excessive blockage	The blocking time should not exceed 10 minutes.
		Inadequate fixation	Choose a proper method for fixation.
		Inadequate antigen retrieval	Increase the period or adjust the system of antigen retrieval. Choose a proper method according to the instruction as well as characteristics of the specimen.
Non-specific staining	Entire tissue is staining	Antibody concentration may be too high	Decrease the concentration of antibody.
		Incubation period is too long	Strictly follow the procedures. Use a timer to avoid long incubation.
		Incubation temperature is too high	Incubation at 4°C is recommended.
		Developing time is too long or the concentration of chromogenic reagent is too high	Developing time should be controlled within 5-10 minutes. It is recommend to monitor it under a microscope in order to stop the reaction in time.
		The tissue has dried out	Keep sections at high humidity to avoid drying.
		Antibody is expired	Using antibody within the expiry date
	Edge of tissue is staining	The edge of tissue do not bound tightly to the slide, so the reagent under the tissue is not washed clearly.	Recut sections thinner (≤ 4 um) Do not use the tissue containing a lot of necrosis areas.
	Edge of tissue is staining	The reagent do not sufficiently cover the section and the reagent around the edge dry out first. The straining of the central is deeper as the concentration of the reagents is higher.	Reagent should sufficiently cover the tissue.
	Only half of the tissue is staining	Reagent do not cover the whole section	Ensure reagents have covered the whole section.
		Staining dish is not kept horizontal, part of tissue is not covered by reagent	Keep the staining dish horizontal.
		Part of tissue can not be stained because there are bubbles on the slide	Exclude bubbles by toothpick
	Patchy staining	The tissue is out of flatness as there is bubbles in the slide. The staining is not uniform caused by dying reagent residue.	Exclude the bubble in the rinsing dish. Keep 45 degree angle in order to remove liquid after washing.
		There are necrosis tissue on the section	Do not choose sections containing a lot of necrosis tissue.
		The concentration of gel is too high when making APES slide	Add adequate acetone if it evaporates.
	Intercellular substance is staining	Non-specific staining caused by the hydrophobic interaction between antibody and hydrophobic group of protein in the tissue.	Block with the serum to avoid non-specific binding before adding primary antibody.
		Non-specific reaction of the antibody	Change the specific antibody
		The antibody is contaminated	Store antibodies in aliquot at -20° for later use. Avoid repeated freeze/thaw cycles and cross contamination.
	Cytoplasm is staining	Endogenous peroxidase activity is present	Use enzyme inhibitors such as H2O2 to eliminate the non-specific staining.
		Several antigen or Fc fragment introduced by Endocytosis of Macrophage.	Identify macrophages by Morphology.
		Endogenous biotin is staining	Block endogenous biotin with egg white. Use non-biotin detection system ( EliVision,EnVision) to avoid the interference.
	Cell nuclear is staining	Tissue is kept too long in the buffer(such as xylene)	Control the soaking time in the buffer.
		The tissue has dried out	Add liquid in time
		Inadequate repair mode	Adjust the factors such as PH, time and repair method.