Immunohistochemistry Staining Tips

For getting qualified immunohistochemistry staining section, we collect and list the tips one may encounter and pay attetion to:

• Fresh fixed tissue

In the final analysis of immunohistochemistry staining, it normally exists of non-specific staining. Actually, it should be due to diffusion of antigens. Take tumor tissues for an example, the inner antigens are most likely to diffuse as a result of the infinity and high-speed of tumor cells’ proliferation. The poor blood-supply in the middle part of tumor tissues causes severe ischemia and necrosis. Antigens in dead cells then disperse into intercellular substance.

Another reason for this fake non-specific staining is the delayed fixation. Autolysis will occur if tissues are not fixed timely.

• Sufficient dehydration of tissues

A selected tissue should not be too thick. Thickness will affect dehydration, paraffin-embedding and sectioning. A suitable thickness of tissue is a necessity for subsequent immunohistochemistry staining result.

• An intact, uniform and smooth sectioning

Smoothness and less air bubble will facilitate adhesion of tissues, and washing steps in immunohistochemistry staining, too.

• Binder materials (This may differ for different slides you purchased)

Newly purchased slides have oil and fat on their surfaces, therefore a treatment for slides is needed. Immerse slides in glass cleaning solution for 4 hours to overnight. Wash with water and immerse in ethanol for 2 hours or longer. Immerse slides in solution of 3-aminopropyl triethoxy-silane:ethanol (acetone)=1:50 for 10 minutes and wash with ethanol for 2 times.

• Sufficient deparaffinization

Due to its hydrophobicity, paraffin is insoluble in antibody solution. An insufficient deparaffinization will lead to uneven immonihistochemistry staining, and enhancing background, too. Normally dimethylbenzene is used to deparaffinize. Typically, at a relatively high room temperature, the deparaffinizing time could be shortened to 3-5 minutes, and at low temperature the time should be lengthened to 10-20 minutes.

• A thorough inhibition of endogenous peroxidase (for immunoenzymological staining such as DAB, HRP)

In many tissues and cells such as red blood cells, neutrophils, monocytes and eosinophils there are much peroxidase. Therefore an endogenous peroxidase inhibition step is needed. A too strong inhibition will bring damage to antigens, thus it’s an inhibition step but not an elimination step. Typically 0.3% H2O2 is used as the inhibition solution.

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