Autoclaving or Pressure Cooking

In order to standardize the procedure, it is important to start with standard volumes of preheated solutions. After adding the specimens to the boiling retrieval solution, the autoclave or pressure cooker should be brought to full pressure as quickly as possible and the heating times measured exactly from this point. At the end of the heating time (usually 1 to 2 minutes) the pressure should be released. As soon as possible the hot buffer should be flushed out with cold water. (Sections should not be allowed to dry.) The specimens should then be washed in buffer.

Although the most critical feature of both microwaving and autoclaving is probably the heating of the tissues, the pH and composition of the solutions used are also important in the unmasking of antigenic sites. Studies have found no significant difference between microwave and autoclave treatment, but there are significant differences based on the solutions used. Some of the buffer solutions commonly used are 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0), with citrate buffer used most commonly. It should be noted that many more specimens can be treated at any one time using an autoclave or pressure cooker than using a microwave oven. However, preservation of the cytological detail may be slightly inferior in sections that undergo pressure cooking. Chemicon provides a recommended HIER protocol on its Technical Service website menu available at: www.chemicon.com/techsupp/protocol/HIER.asp

A more mild procedure that can be used on many tissues is a simple incubation in citric acid buffer, pH 3.0 (2.1 grams Citric Acid added to 400 mL of ddH₂0. Adjust to pH 3.0 with Acetic acid if above 3.0, or NaOH if below 3.0, make up to 1 L final volume with ddH₂0) for 30 minutes, at 37篊 after blocking but prior to primary antibody addition. Rinse slide in PBS or TBS pH 7.4 prior to staining.

Antibody Staining:

Primary antibody may be directly labeled with an enzyme (such as horseradish peroxidase or alkaline phosphatase) or fluorophore (such as FITC or rhodamine), or unlabeled, with detection by a labeled secondary antibody or more complex detection system. If a secondary antibody is used, it must be generated against the immunoglobulins of the primary antibody source, e.g., if the primary antibody is raised in rabbit, then the secondary antibody could be goat anti-rabbit. The optimal titer of both the primary and secondary antibody should be determined for each batch.

The proper working dilutions for every antibody must be optimized for the system in which it is being employed. The same system does not always work for every antibody. Antibodies are like children, and each is different. The product data sheets may be used as a guide for dilution series starting points. (See Appendix B for a possible dilution protocol.) The optimal antibody dilution will be that which gives the strongest specific antigen staining with the lowest non-specific background. As with other controlled experiments, it is advisable to change only one experimental variable at a time. After determining the optimum titer/dilution of the primary antibody, the secondary antibody dilution can be optimized.

For staining of tissue sections, it is customary to incubate with 25?0 礚 of diluted antibody ?the volume used must be sufficient to completely cover the tissue, and to ensure the tissue will not dry out during incubation. Incubation times may range from 30?0 minutes at 37癈, from one to six hours at room temperature, or overnight at 4癈. Incubation times should be optimized empirically for each antibody/antigen combination.

Chemicon抯 IHC Select^{?/sup> Manual Staining System (Cat. No. MSS001) offers a convenient platform for quality antibody staining. This compact device offers the convenience of standardized staining with the efficiency of capillary gap technology to reduce reagent volume and minimize waste.}

General Protocol for Immunohistochemical Staining with Polyclonal Rabbit or Monoclonal Mouse Primary Antibody:

The following general protocol is intended for use as a guideline in developing antibody-specific procedures. Different antibodies and tissues may require changes to this procedure. Review of individual product datasheets and relevant literature references may be helpful in customizing this procedure for specific applications.

1. Gently rinse slide containing sections with distilled water or buffer from a wash bottle. Place slide in room temperature buffer bath for 5 minutes to rehydrate sections.
    
2. Using a Kimwipe^{?/sup>, gently remove excess liquid from around the specimen. Avoid touching the tissue directly.}
    
3. Apply 4? drops of normal serum, (normal serum from the host of the secondary antibody), diluted 1:5?:30 (final conc. 3%?0%). Incubate for 20?0 minutes at 37癈.
    
4. Tap off serum and wipe away excess. Do not rinse.
    
5. Perform any antigen retrieval if necessary.
    
6. Apply 25?0 礚 of rabbit (mouse) primary antibody, diluted appropriately, per tissue section. Antibody should cover sections completely. Incubate for desired time (see above for suggested parameters and temperatures). If optimal antibody dilution is unknown, perform a series of antibody dilutions in the range of 1:20?:1,000 to obtain initial results.
   
   Note: Antibody diluent is often very important for consistent reactivity. Simple solutions are easier to troubleshoot then complex ones, thus antibodies diluted only with simple buffers (PBS or TBS) are usually recommended.
    
7. Rinse slide gently with distilled water or buffer from a wash bottle, and incubate in a buffer bath for 3 x 5 minutes (changing buffer in between washes).
   
   Note: For all procedures it is important to see that each step is adequately buffered, and that non-reacted solutions are washed away after each step.
    
8. Apply 25?0 礚 of enzyme-conjugated antibody directed against rabbit (mouse) immunoglobulins, diluted appropriately. Incubate 45?0 minutes.
    
9. Rinse slide gently with distilled water or buffer from a wash bottle, and incubate in a buffer bath for 3 x 5 minutes (changing buffer in between washes).
    
10. Apply substrate-chromogen solution and incubate until desired color intensity has developed.
     
11. Rinse gently with distilled water from wash bottle. Counterstain and coverslip.

Kimwipe^{?/sup> is a registered trademark of the Kimberly-Clark Corporation}

Antibody Detection:

• Enzyme-Mediated
• Fluorescence
• Signal Amplification

Two of the most commonly used detection methods are fluorescence and colorimetric (enzyme mediated) detection. With the advent of electron microscopy, detection of antigens by antibodies that contain large gold particles is often used, and these may also be visualized at the light microscopic level as well, but their use is quite rare today, outside of electron microscopy. Described below are the common antibody detection methods for light microscopy.

Enzyme-Mediated Detection

When choosing a substrate for conversion by an enzyme, one should select a substrate which yields a precipitating product. Examples of commonly-used substrates are listed below.

Fluorescence

A molecule that fluoresces can be attached to the antibody for detection using UV light. Examples are Fluorescein, Rhodamine, Texas Red? Cy3 and Cy5. In selecting fluorochromes, one is limited by the available microscope filter sets. Most filter sets are best matched with rhodamine or fluorescein. Texas Red^{?/sup> may also be used with a rhodamine filter set.}

Many mounting media contain 揳nti-fading?solutions, such as DABCO, which will prolong the viewing time of the sample. Chemicon offers a variety of fluorescence mounting fluids and counterstain solutions including our basic fluorescent mounting fluid (Cat. No. 5013) and enhanced counterstaining fluid containing nuclear stains such as DAPI and PI (Cat. No. S7113, S7114).

Texas Red^{?/sup> and Alexa Fluor?/sup> are registered trademarks of Molecular Probes, Inc.
Fluoro-Jade?/sup> is a registered trademark of Histo-Chem, Inc.}

Signal Amplification

Signal amplification techniques greatly enhance the sensitivity of immunohistochemical and immunocytochemical methods. The signal amplification methods may be used in conjugation with either of the above detection techniques. Signals may be amplified by using poly-conjugated secondary antibodies (i.e. Chemicon catalog numbers AP340P朅P342A series), or Avidin-Biotin interactions or other commercially-available amplifiers (i.e tyramide catalyzed systems), which increase the signal to antibody ratio. When signal amplification is used to amplify the specific signal, however, one should be aware that non-specific signals may also become amplified. Thorough washing and proper antibody titration is especially important in this case.

Troubleshooting:

• No Staining
• Weak Staining
• Background Staining

When tissue staining has not given the expected results, the experiment should be examined in a systematic way, wherein only single experimental variables are altered at one time. Proper immunohistochemical troubleshooting requires one to determine whether difficulties are related to specimen, antibodies, technique, environment, or slide interpretation? The following checklist may assist in troubleshooting efforts.

No Staining of Either Controls or Specimen

• Confirm that no reagents were omitted (primary antibody, secondary antibody, substrate components)
   
• Confirm that reagents were added in the correct order, and for sufficient incubation times.
   
• Re-read labels to confirm that correct antibodies were used. This is especially important when using primary/ secondary antibody combinations. For example, when using a mouse IgM primary antibody, the secondary antibody should be a goat or rabbit anti-mouse IgM (not IgG).
   
• Check antibody titrations and dilutions. This is particularly important for the primary antibody.
   
• Check that antibody blocking solutions or diluents do not contain substances that may interfere with or absorb the primary antibody. For instance, serum containing diluents can absorb out or reduce primary reactivity to small molecules such as GABA or amino acids.
   
• Check reagent expiration dates and storage. Enzymes and fluorochromes are especially prone to breakdown after prolonged storage. Antibodies should be stored in nondefrosting freezers, as self-defrosting models will expose antibodies to repeated freeze/thaw, resulting in antibody breakdown.
   
• Check specimen storage. Where possible, compare staining of the unknown specimen versus a known positive tissue in a 搒ide-by-side?experiment.
   
• Check the chromogen/substrate solution. This can be checked by adding a drop of the labeling reagent to a small sample of prepared chromogen. If the chromogen is working, the mixture should change color. Chromogen solutions can deteriorate quickly, so do not use beyond the times recommended by the manufacturer. Lack of color change may be due to inactive enzyme or improperlyprepared chromogen.
   
• The rinse buffer may be incompatible with the reaction reagents. The pH must be appropriate, and buffers to be used with peroxidase enzyme should not contain NaN₃.
   
• The counterstain and mounting media may not be compatible with the chromogen. Check the manufacturer抯 recommendations.
   
• Check that the microscope is adjusted correctly and that the fluorescence lamp is not burned out.

Weak Staining

Points to consider are:

1. Is the intensity of the staining consistent between the positive controls and the test sample(s)?
    
2. Is the staining specific for the antigen of interest, or is it background staining? This can only be determined by examining the slides.

All the items listed above for No Staining can apply to a lesser degree to the situation of weak staining. However, if the negative controls are devoid of stain and the positive controls and test sample(s) are weakly stained, then possible trouble points include:

• Overfixation, or incorrect fixation for the immunological procedure in use
   
• Insufficient antigen retrieval
   
• Antibody concentration may be too dilute. If possible, the concentration should be increased. If this is not feasible, then the incubation time or temperature may need adjusting. When diluted antibody is stored in the refrigerator it sometimes gets absorbed to the walls of the container. Storing the antibody with a protein carrier such as 1%?% BSA can alleviate this situation.
   
• Too much buffer rinse has been left on the slide, so that the antibody becomes diluted when added to the sample.

If the negative controls have not reacted, the positive controls are well stained, but the test sample is stained weakly, then either the positive control and the test sample were fixed differently, are of different tissue type, or the outer tissue of the test specimen block has been poorly fixed.

If the negative controls have not reacted, the test sample(s) are well stained, but the controls are weakly stained, then the control material should be replaced.

Background Staining

If the negative control is being stained as well as the positive controls and test sample(s), then the degree and type of background staining must be analyzed. The following are possibilities for investigation:

• Re-titer antibodies (both primary and secondary) with a dilution series.
   
• Incubate with chromogen for a shorter time. Some chromogens, such as DAB, develop very quickly.
   
• The chromogen was not totally dissolved, and associated with the tissue. Centrifuge or filter the chromogen solution.
   
• Particulates in the antibody solution. These may form upon repeated freeze/thaw and can be eliminated by centrifugation.
   
• Insufficient rinsing between steps, or contaminated buffers. Mix new buffers and increase washing steps.
   
• Enzyme or biotin in the tissue is reacting with the reagent. This can be prevented by increasing the time or concentration of block, trying different types of block, or using a combination of more than one block, or changing the staining methods. Some tissues (i.e. brain and liver) are known to contain high endogenous levels of biotin or peroxidase activity.
   
• The incorrect blocking serum was used, or blocking serum was not used. The blocking serum should be from the species of the secondary antibody. It is possible to use 5% nonfat dry milk rather than serum.
   
• The secondary antibody cross-reacts with endogenous tissue proteins. Secondary antibodies which have been absorbed against immunoglobulin from the species from which the target tissue was obtained will result in significantly lower background, and are indespensible for double-labeling experiments.
   
• Hydrophobic and/or ionic interactions between the reagents and tissue types such as connective, adipose or fatty tissues may give rise to apparent specific reactions. Antigen retrieval procedures can be of great assistance in correcting this predicament. A decreased fixation time in formalin can also help.
   
• The embedding media may not be completely removed from the tissue. Review the removal procedure for possible changes.
   
• The specimen may have dried out during the procedure, allowing the trapping of reagents under the edges of the specimen. Care should be taken to avoid letting the specimens dry.

If there is background staining in the positive controls and the test sample(s), but not in the negative control, then the issue is most likely associated with the primary antibody. Some possibilities are:

• The primary antibody was too concentrated, or the incubation period too long. More dilute antibody, or shorter incubation or lower incubation temperature may correct the situation.
   
• The tissue may contain Fc receptors, or there may be interfering Ig components (aggregates or oligomers) or there may by naturally occurring, contaminating antibodies. This can be resolved by using Fab fragments rather than whole IgG molecules, filtering out the aggregates, or by diluting the primary antibody and incubating for longer times.
   
• The tissue sections may be cut too thick ?try thinner sections.
   
• The microscope light needs to be adjusted to a higher setting.

If there is background staining in only the test sample(s) - i.e. not in the positive or negative controls, then the most likely cause is that the test sample(s) has been fixed and processed differently from the controls. Use of different tissue type between test sample(s) and controls may also produce this variance. Possibilities include:

• Overfixation of the test sample, resulting in the increased presence of hydrophobic groups, or increased crosslinking. Use of antigen retrieval procedures will amend this.
   
• A different fixative was used for the test sample(s) than for the control tissue. This difference should be avoided, or the procedures should be adjusted.
   
• The test sample(s) and the controls are of different tissue type. This should be avoided whenever possible.

If the test sample(s) and positive controls are clean, but the negative control shows background staining, it is likely that the negative control serum is at fault. It may be too concentrated, or contaminated with cross-reacting Ig components, naturally occurring antibodies, or bacterial growth. This can be corrected by using more dilute serum and incubating longer, trying to find a better match for the negative serum, or purifying the serum.