Step 73 - Use High Quality Sections
Take particular care to use thin, flat sections that have been thoroughly dried onto the slide. Preferably use charged slides or APES coated slides for IHC.
Uneven, poorly-adhering sections stain unevenly with variable background staining.
A A bubble under the section (from mounting) has resulted in subsequent detachment of the section during staining (tonsil, CD45).
B A poor quality section, that has not been properly flattened and dried before staining, has lifted making the slide unsatisfactory (tonsil, CD3).
 |
Step 74 - Ensure Optimal Fixation
Good quality fixation using known and consistent fixation conditions (fixative type, pH, temperature, time) produces the best results. Specimens should be checked prior to processing to determine if further fixation is required.
Inconsistent fixation conditions, producing under-fixed or over-fixed tissues, produce variable results and make troubleshooting difficult.
Uneven fixation (zonal fixation) has resulted in uneven staining in this section (breast tumor, ER).
 |
Step 75 - Avoid Section Adhesion Problems
Avoid the use of protein-based section adhesives in the flotation bath (glue, starch, orgelatin), particularly on charged slides.
Protein-based adhesives can block the surface of the charged slide. This causes inconsistent adhesion and leads to uneven staining due to pooling of IHC reagents beneath lifting sections.
A line of thick protein-based section adhesive has stained adjacent to the section (breast, PR).
 |
Step 76 - Optimize Wax Removal and Reagent Application
Take particular care with dewaxing and hydration of sections as well as efficient and uniform distribution of reagents on the specimen surface. This ensures even staining and consistent results.
Incomplete removal of wax or uneven distribution of reagents on the specimen surface can produce unstained or poorly stained areas in sections.
A Poor reagent flow has produced uneven staining (tonsil, CD45).
B Residual wax has resulted in an unstained area (tonsil, CD5).
C A bubble in the primary antibody has prevented uniform staining (tonsil, CD20).
 |
Step 77 - Avoid Concentration Gradients
Concentration gradients are avoided by careful application of reagents.
“We sometimes see strong staining at one end of the slide progressing to weak staining at the other.”
A and B are micrographs taken from opposite ends of the same slide. One end of the slide shows strong staining (A) where as at the other end (B) the staining was very weak. This is an extreme example of a concentration gradient created during staining (tonsil, CD45).
 |
Step 78 - Choose Antibody Carefully
Choose your primary antibody carefully with regard to its sensitivity and specificity. Be aware that antibodies sold by different suppliers often come from the same source and are repackaged/branded for sale. It is important to use the clone name when assessing an antibody.
“We buy our antibodies based on price alone.”
These sections of human tonsil from the same block have been stained with the B cell marker CD20 using primary monoclonal antibodies from different sources (suppliers). In each case the recommended pretreatment and optimized dilution was used. There is an obvious difference in the quality of the results achieved.
 |
Step 79 - Read Specification Sheets
Know your primary antibody. Always check the specification sheet to determine the suitability of your method for a particular antibody. Specification sheets should be updated when a new batch of antibody is purchased.
“We don’t have access to the antibody specification sheets in our laboratory.”
These sections of intestine have been stained for Cytokeratin AE1/AE3. Different retrieval conditions were used for each section. Section A shows unacceptable weak staining, while section B shows strong precise staining.
 |
Step 80 - Optimize Retrieval Methods
Choose appropriate unmasking conditions for the primary antibody being used, the tissue being stained and the fixation employed (pH, reagent, reaction conditions).
The same retrieval technique is used for all primaries on the assumption that there is a successful universal HIER method.
Prostate sections stained for Cytokeratin 34βE12. Section A shows weak staining while section B is stronger and sharper. The only difference between the two was the retrieval method used.
 |
Step 81 - Consider Antibody Cross-reactivity
Be aware of any potential problems with antibody cross- reactivity (read the specification sheet).
No attempt is made to explain unexpected positive staining.
Palatine tonsil showing the base of a tonsillar crypt stained for CD5, a lymphocyte marker that stains mainly T cells. This particular clone (4C7) cross- reacts with epithelial cells deep in the crypt.
 |
Step 82 - Block Endogenous Peroxidase
For peroxidase-based detection systems, always use a peroxidase-blocking step.
Non-specific staining is often seen in erythrocytes, granulocytes, monocytes, and in muscle. This is due to incompletely-blocked endogenous peroxidase.
Spleen showing typical, non-specific staining of erythrocytes due to incomplete blocking of endogenous peroxidase. Here the natural peroxidase present in the red cells has reacted with the DAB chromogen.
 |
Step 83 - Avoid Background Staining
Appropriate protein block is always used.
Generalized background staining is sometimes seen due to ineffective protein block.
Normal tonsil stained for Kappa light chain showing a heavy back-ground stain due to ineffective protein block.
 |
Step 84 - Use an Appropriate Detection System
Choose an appropriate detection system that will provide precise, specific staining with adequate sensitivity.
“We have been using the same detection system for a long time and see no reason to change. Sometimes our stains are weak and are not as sharp as we would expect.”
Sections A and B are from the same specimen but have been stained using different detection systems. Note the difference in the intensity and precision of the stains. (tonsil, CD20).
 |
Step 85 - Standardize Washing Steps
Use standardized washing steps throughout (duration, volume and form of agitation). This will ensure consistency of results.
Results are very variable within runs with the same antibody and between runs on different days. This can be due to different washing techniques used by different operators.
Sections A and B are from the same specimen and have been stained manually using the same reagents. Note the difference in the level of background staining in the stratified epithelium. This is probably due to a difference in the efficiency of the washing technique used. (tonsil, CD20).
 |
Step 86 - Optimize Counterstaining
The level of nuclear counterstain is carefully regulated and standardized so as not to obscure positive staining. The counterstain should provide the best possible contrast between chromogen and background tissue elements. An appropriate counterstain is chosen for the chromogen used.
Nuclear counterstain is sometimes very strong. This can obscure weak specific staining.
Tonsil stained for Ki67, a nuclear marker for proliferating cells. The two sections are from the same specimen showing different levels of hematoxylin counterstaining. Slide A shows staining that is too strong and would obscure a weak positive reaction. Slide B shows a better level of staining.
 |
Step 87 - Use Appropriate Controls
Always use appropriate positive and negative controls that are carefully examined to validate results. Internal positive and negative controls are also important and provide an excellent means of ensuring quality assurance in IHC.
“We only do controls when our method doesn’t seem to work. If we did them for every run people wouldn’t bother to look at them.”
Tonsil stained for Ki67. This was the negative control slide and the nuclei should not be stained. The primary antibody was mistakenly applied to this slide instead of the negative control reagent.
 |
Step 88 - Evaluate Results Carefully
Know what to look for and where to look when evaluating your test sections and controls after staining.
If staining is observed in test sections it is assumed the stains are satisfactory.
Intestine stained for AE1/AE3. Unexpected weak staining of the crypt epithelium has occurred. On investigation it was found that CK20 had been wrongly used as the primary antibody.
 |
