Immunostaining (IHC Staining)

Immunohistochemistry (IHC)-staining method:

  • • According to different biotins conjugated with antibodies, IHC staining methods can be classified as immunofluorescence, immunoenzymological staining and affinity histochemistry.
  • • According to different kinds of procedures, immunohistochemistry staining can be divided into subtypes of direct staining (one-step staining) and indirect staining (two-step, three-step or multi-step staining).

Immunohistochemical Staining (Immunofluorescence):


Immunofluorescent staining is the first immunohistochemical staining method. With fundamentality of antigen-antibody binding reaction, antigens are visualized by fluorescence dyes conjugated with antibodies when being activated by exciting light of specific wavelength under fluorescence microscope. Due to high sensitivity, specificity and convenience, immunofluorescence staining method has been widely used in medical analysis and diagnosis.

Immunohistochemical Staining (Immunoenzymological staining):


In immunoenzymological staining enzyme-labeled antibodies are used to bind specific antigens in tissues samples or cultured cells. After adding in substrate of enzyme it generates insoluble or high-electronic density particles that could be localized under light microscope or electronic microscope.

Compared to immunofluorescence staining, immnuenzymological staining has more accurate localizing, better contrast ratio, and samples are able to be stored for a long time.

Immunohistochemical Staining (Immunocolloidal gold technique):


Immunocolloidal gold technique is a kind of technique that uses colloidal gold as a marker. This kind of special metal particle is hydrosol form of gold and it can bind proteins rapidly and stably. Moreover, colloidal gold has little effect on biological activity of natural proteins. Therefore colloidal gold is an ideal material which can be conjugated with primary, secondary antibody or some other kinds of proteins that can bind IgG (e.g. Staphylococcus A protein) to localize certain antigens in sectioned tissue or cultured cells. Due to differences in size of particles of colloidal and its high electronic density, immunocolloidal gold technique is suitable for single or multi-label detection under immune-electron microscope, and light microscope,too.

Learn more about:

Immunohistochemistry (IHC)-staining protocol

Immunohistochemistry (IHC)-staining antibody

Immunohistochemistry (IHC)-staining tips

Immunohistochemistry (IHC)-staining reference

Immunohistochemistry (IHC) Double Staining

Immunohistochemistry double staining technique can be used to visualize two or more antigens and analyze the relationship of their localization, morphology and functions.

Continuous sections double staining

Continuous sections staining may be the simplest way to visualize two antigens. As shown in the name, it respectively stains two antigens on two adjacent sections. Therefore it effectively prevents interference and the fake positive results. However, the two adjacent sections might differ in some aspects such as cell sizes, types and numbers. Repeated experiments are needed to diminish the standard deviation.

Immunofluorescence double staining

IF double stain

Direct staining:

Incubate the sections with mixture of two primary antibodies which are respectively conjugated with two fluorescence dyes (e.g. FITC and TRITC)

Or, successively incubate sections with two primary antibodies;

When processing microscopy two wavelengths of exciting lights should be set and merged picture could be synthesized by image software.

Indirect staining:

In the indirect immunofluorescence double staining the primary antibodies are without fluorescence dyes. Incubate sections with one kind of primary antibody and corresponding secondary antibody, then the other. Process microscopy and get pictures merged by image software.

Immunoenzymological double staining (double enzyme recommended):

PAP double staining

Incubate sections with two primary antibodies;

Incubate sections with corresponding secondary antibodies which are conjugated with two different enzymes (e.g. HRP, AKP), or anti-HRP (PAP complex), anti-AKP (APAAP complex).

Note: *HRP antibody should be incubated prior to that of AKP.

*Perform pre-experiment to prevent the cross-linking reaction.