Immunohistochemistry (IHC) is a method to detect antigen or hapten in tissue section cells by using the specific combination of antibody and antigen in biological tissue. IHC is an important application of monoclonal and polyclonal antibodies, which can be used to determine the tissue distribution of antigens of interest in health and disease. It is widely used in cancer diagnosis because specific tumor antigens are de novo expressed or up-regulated in some cancers. In addition, IHC plays an important role in pathology, especially in tumor pathology, neuropathology and blood pathology.

Figure 1．Illustrations of skeletonized microglia with a circular soma (suboptimal) versus a single origin soma (optimal) and the corresponding overlay between the skeletonized cell and the original photomicrograph (Young, et al., 2018).

IHC uses immunological and biochemical techniques to examine different tissue sections by using labeled antibodies that interact with the target antigen. CD BioSciences' multiple antibody staining schemes can produce informative IHC images, allowing visualization of the unique cellular components of cells. If you have any needs, please feel free to contact us.

Immunohistochemistry technology includes two stages: 1) the stage of slide preparation and reaction involved, 2) the stage of interpretation and quantification of the obtained expression. The five key steps of IHC are preparing sample, retrieving antigen, blocking background, detecting target, and visualizing sample. Detailed tips and techniques for IHC are as follows.

Guidelines for Choosing a Fixative

Antigen	Fixative
Most proteins, peptides and enzymes of low molecular weight	Cells / cytological preparations: 4% formaldehyde
	Tissue sections: 10% Neutral-Buffered Formalin (NBF)
Delicate tissue	Bouin's fixative
Small molecules such as amino acids	4% formaldehyde
Blood-forming organs (liver, spleen, bone marrow)	Zenker's solution
Connective tissue	Helly's solution
Nucleic acids	Carnoy's solution
Large protein antigens (e.g., immunoglobulin)	Ice-cold acetone or methanol (100%)
Nuclear morphology	Zinc formalin
For electron microscopy	4% formaldehyde - 1% glutaraldehyde

Possible Causes of No Staining in IHC

Possible causes	Solutions
The primary antibody and the secondary antibody are not compatible	Make sure you use a secondary antibody that was raised against the primary antibody species.
Not enough antibody is bound to the protein	Add a higher concentration of primary antibody
	Incubate the sample for longer with the antibody at 4°C.
Antibodies or amplification kits may have lost activity due to improper storage and handling	Check the storage instructions for your products on the datasheet.
	Avoid excessive freezing / thawing.
	Run a positive control.
The protein of interest isn't present in the tissue	Run a positive control.
	Check the scientific literature to see if protein is expected in the tissue type.
The protein of interest is present in low abundance	Use signal amplification to maximize the signal, eg a biotin-conjugated secondary antibody.
The fluorophore may have been damaged by too much light exposure	Always keep fluorophore-conjugated secondary antibodies in the dark; exposure to too much light can lead to photobleaching.
Deparaffinization may be insufficient	Deparaffinize sections longer and use fresh xylene.
The buffer is contaminated with bacteria	Add 0.01% azide to the antibody storage buffer.

Application of Immunohistochemistry

1. Duraiyan, Jeyapradha, et al. "Applications of immunohistochemistry." Journal of pharmacy & bioallied sciences 4.Suppl 2 (2012): S307.
2. Young, Kimberly, and Helena Morrison. "Quantifying microglia morphology from photomicrographs of immunohistochemistry prepared tissue using ImageJ." Journal of visualized experiments: JoVE 136 (2018).

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