Immunohistochemistry / Immunocytochemistry Procedures

In these techniques an antibody is used to link a cellular antigen specifically to a stain that can be more readily seen with a microscope. Detection of antigens in tissues is known as immunohistochemistry , while detection in cultured cells is generally termed immunocytochemistry . For both, there is a wide range of specimen source, antigen availability, antigen antibody affinity, antibody type, and detection enhancement methods. Thus optimal conditions for immunohistochemical or immunocytochemical detection must be determined for each individual situation, dependent on the above variables. As for all procedures, reference should be made to individual product data sheets and published literature. Also, the Internet today contains a tremendous amount of useful information on Immunohistochemistry/ immunocytochemistry. Quick searches through google.com or similar search engines are highly recommended.

Specimen Preparation :

• Fixatives
• Section Type

Fixatives :

Fixatives are needed to preserve cells and tissues in a reproducible and life-like manner. To achieve this, tissue blocks, tissue sections, cell growths or smears are either immersed in a fixative fluid, or in cases where whole animal systems are studied, the animal is perfused with fixative via its circulatory system (For perfusion protocol, see www.chemicon.com/techsupp/Protocol/perfusion.asp ). In the case of cells in culture, cell preparations are either submerged or simply air-dried. Fixatives stabilize cells and tissues thereby protecting them from the rigors of subsequent processing and staining techniques. For immunological studies, fixation is especially imperative to ensure the adequacy of the specimen and target antigens. Tissues have differing protein content and structural arrangement, thus they have a variable ability to retain their structure without significant fixation. Incorrect specimen preparation can block or impede antigen labeling in tissue and cells. Unfortunately, the methods that are best for the preservation of tissue structure do so by altering proteins, thereby masking some epitopes, and sometimes preventing the detection of the desired target protein. In cases of failure it is important to experiment with multiple different fixatives and antigen retrieval methods prior to �iving up?on a specific stain.

Fixatives may work by several means: formation of crosslinkages (e.g., aldehydes such as glutaraldehyde or formalin); protein denaturation by coagulation (e.g., acetone and methanol); or a combination of the above. Fixation strengths and times must be optimized so that antigens and cellular structures can be retained and epitope masking is minimal. Requirements for fixation can vary widely between tissues. For example when using antibodies to probe for neurotransmitter substances, most tissues must be either immersion fixed with a mixture of glutaraldehyde and paraformaldehyde, or with paraformaldehyde alone. Both acetone and methanol (precooled to -20�) have been used successfully as fixatives for frozen tissue in other instances.

Immunocytochemistry

Fixation of cultured cells for immunocytochemistry also requires careful consideration; in general, fixation strengths and times are considerably shorter for cells than on the thicker, structurally complex tissue sections. For immunocytochemistry, sample preparation essentially entails fixing the target cells to the slide. Depending upon the needs of the experiment, cells can be simply harvested and �ropped?on to a slide, fixed and air-dried, as is often done with clinical or diagnostic studies where speed and a simple �es/no?answer is all that is needed. Alternatively, where information on the structural location within the cell is needed, cells are often cultured directly on prepared slides or coverslip material, which are then simply washed and fixed in place prior to staining.Consulting published literature relating to the tissue/proteins of interest is well worth the time invested. See Appendix A for recipes of common fixatives.

Section Type:

• Cryostat (frozen) sections
• Paraffin (wax) sections

The next consideration for immunological staining is the type of section to use. For immunohistochemistry, the common options are fixed or unfixed cryostat (frozen) sections, fixed �et?or vibritome sections, or fixed, paraffin-embedded sections. The choice of section is determined by a number of issues, including the time and skill of the investigator. Because of the ease of use, fixed frozen sections are often quickest and easiest to use. However, because of their superior fidelity, clarity, and preservational properties, fixed paraffin-embedded tissues have become the ultimate standard of immunohistochemisty in histology and pathology, and anytime where archiving of immunohistochemical information is required.

Cryostat (frozen) sections

Today there are two types of Cryostat sections: (1) Fresh, or unfixed sections where quickly frozen (snap frozen) tissues are first cut, then either air-dried or fixed prior to staining; (2) or fixed frozen tissue, where the tissue is first fixed then cryoprotected with sucrose or other stabilizer (to stabilize the tissue cell structure) prior to freezing and sectioning. The advantages of frozen sections are that they allow excellent antigen preservation, they are typically faster to perform, and they offer flexibility, since any fixative can be used, thereby facilitating the optimization of fixative for each antigen. However frozen sections give less morphological detail and resolution than other methods.

Sample Protocols for Cryostat Sections - Fresh Frozen (then fixed) Tissue Sections:

1. Snap-freeze small tissue blocks (5x5x3 mm) in liquid nitrogen.
    
2. Transfer to cryostat and cut thin (5?0 �) sections.
    
3. Collect specimens on clean poly-L-lysine-coated glass slides and dry at room temperature overnight (if you want to stain the same day let air-dry for 1? hrs. until completely dry). Thorough drying is essential for good adhesion to the slides.
    
4. Fix sections in acetone or absolute ethanol at 4� for 15 min. Use fresh ethanol or acetone for every 10?5 slides for best results. The organic solvents absorb moisture from the air and tissue, as they do so, they lose their ability to fix the tissue effectively.
    
5. Thoroughly air-dry at room temperature or on mild heat (30?7�). It is during this stage that much of the chemical fixation is being finalized; improper air-drying will lead to �oft?sections and likely loss of proper reactivity.
     
6. Proceed with immunostaining or freeze.

Fixed, Frozen Tissue Sections:

1. Fix tissue either by perfusion with fixative or by immersion in fixative for a set time period. Most commonly, 4% Paraformaldehyde (PFA) solutions are used.
    
2. Fixed tissue is then prepared for cryoprotection by submerging the target tissue in a hydrostabilizing solution. The cryoprotection is complete when the target tissue no longer floats in the stabilizing solution. Because it works well and is relatively inexpensive, PBS+sucrose solutions ranging from 10% (less protection), to 30% (w/v) sucrose (greater protection) are often used.
    
3. Once stabilized, tissues can be removed from the protectant solution and frozen at -70� until sectioned.
    
4. Sectioned via cyrostat (5?0 �~B~E_where_sections_can_be_collected_directly_onto_slides~E_or_floated_onto_slides_via_a_PBS~Hwaterbath._Usually_up_to_3_sections_per_slide_can_be_placed~J_each_spaced_well_apart._The_spacing_prevents_reagent_mixing_between_samples._*Individual_skill_and_tissue_type_will_determine_the_thickness_of_the_sections._Sections_between_10~N5_� provide the best results for clarity and integrity. Sections between 6? � tend to tear during cutting, resulting in rough edges that can increase the background. Thicker sections while stronger during handling can be more difficult to stain.
    
5. Sections on slides are thoroughly air/warmed dried on a slide warmer, usually overnight or at least 2? hrs. at 40?0�.
    
6. Prepared slides can be stored dry at -70� until stained. Equilibrate to room temperature and briefly redry prior to rehydration and staining.

Paraffin Sections

The largest proportion of samples used in immunostaining are embedded in paraffin because it provides for excellent morphological detail and resolution. Modern �araffin?is typically a mixture of paraffin wax and resin. It is an excellent embedding medium because it can be heated to liquid state, and dissolved by xylene for infiltrating the tissue, and then relatively quickly turned to a solid state again for maximum structural support during sectioning. Typically, small blocks (10x10x3 mm) of tissue are fixed for up to 24 hrs. The most common fixatives used in paraffin sections are formalin-based. These fixatives are well tolerated by the tissues and achieve good penetration. See Appendix A for recipes of common fixatives. The blocks are then infiltrated and embedded with paraffin and 5?0 � sections are cut in ribbons and mounted on slides. Once mounted, the slides can be stored indefinitely until immunostaining is required; then the paraffin must be removed from the tissue to allow the water-based buffers and antibodies to penetrate.

Sample Protocol for Paraffin-embedded Sections:

A. Conventional deparaffinization and dehydration sequence:

1. Incubate sections in Xylene: 2 to 3 changes, 5 min. each.
2. 100% absolute ethanol: 2 changes, 3 min. each
3. 95% ethanol: 2 changes, 3 min. each
4. 80% ethanol: 3 min.
5. 50% ethanol: 3 min.
6. Rinse with distilled water, PBS, or Tris buffer: 2 changes, 3 min. each.

Note: Once sections have been rehydrated, do not allow them to dry.

B. Place slides in prewarmed (37�) 0.1% trypsin in PBS for 5?0 min. or 0.4% pepsin in 0.01N HCl for 30 min. to one hour. Follow by rinsing with distilled water

C. If peroxidase conjugate is used, endogenous peroxidase should be blocked at this stage. Peroxidase activity results in the decomposition of hydrogen peroxide (H ₂ O ₂ ). It is a common property of all hemoproteins such as hemoglobin, myoglobin, cytochrome and catalases. Suppression of endogenous peroxidase activity in formalin-fixed tissue entails the incubation of sections in 3% H ₂ O ₂ for 8?0 min. Methanolic H ₂ O ₂ treatment (1 part 3% H ₂ O ₂ plus 4 parts absolute methanol) for 20 min. can also used, but it is not recommended for specimens where cell surface markers are to be stained. Methanolic treatment may also detach frozen sections from their carrier glass.

D. Wash twice with PBS.

E. Proceed with immunostaining procedure (see Antibody Staining section).

Antigen Retrieval :

• Enzymatic Digestion

• Microwave

• Autoclaving or Pressure Cooking

To facilitate the immunological reaction of antibodies with antigens in fixed tissue, it may be necessary to unmask or �etrieve?the antigens through pretreatment of the specimens. There are many forms of antigen retrieval (sometimes called antigen recovery), and different antigens and different antibodies will require different antigen retrieval methods. Antigen retrieval has been shown to increase reactivity of the majority of antigens in tissues. The use of antigen retrieval in immunocytochemistry is less common, however depending upon the particular antibody/antigen combination it can be performed on cell preparations, although the length of time and intensity is typically much less than for tissue.

Antigen retrieval includes a variety of methods by which the availability of the antigen for interaction with a specific antibody is maximized. The most common techniques are enzymatic digestion or heat induced epitope retrieval (HIER) through microwave irradiation, autoclaving or pressure cooking.

Enzymatic Digestion

This technique involves dewaxing, rehydrating, and rinsing the specimen in running water. The specimen is then equilibrated with the appropriate buffer, and incubated with a proteolytic enzyme at 37�, or at room temperature. Enzymes used include pronase (0.05% (w/v) in PBS), trypsin (0.05% (w/v) in PBS with 0.1% CaCl ₂ ) and pepsin (0.05% (w/v) in 2 N HCl). The conditions of concentration, time and temperature must be controlled, so that the enzymes can break some of the bonds formed during fixation, uncovering antigenic sites, but the antigen should not be digested completely. The enzymatic activity is stopped by placing the specimen in cold buffer (4�) prior to processing with antibody. These methods should be considered for some antigens/tissues. (Shi, S-R et al. (1993). J. Histochemistry & Cytochemistry 41 :1599?604). However, proteolytic enzymes can abolish the reactivity of some antigens. (Pileri, S. et al. (1997). J. Pathology 183 : 116?23).

Microwave Irradiation

Microwave irradiation of formalin-fixed, paraffin-embedded specimens in buffer has been found to markedly enhance the retrieval of antigens. During this procedure the energy provided helps break some of the bonds formed during fixation, thus increasing the number of positive cells available, and the intensity of reactions, although the exact mechanism is unclear. It is important to monitor the sections during the microwaving process, to prevent damage and drying. Consistency of conditions between experiments, including buffer volumes, irradiation times, and microwave unit used, will result in less variability in staining results. The number of samples that can be treated by microwave irradiation at one time is limited. Typically specimens in some buffer (see below) are heated either at full or partial power for a few minutes. Periodically the heating is stopped and liquid is replenished. After a set time, the solution containing the slides is allowed to cool to room temperature slowly, then the slides are rinsed in PBS and used for staining.

 

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