Poor staining continues to be one of our most frequently encountered problems in the histology lab. In this technical letter we address various reasons for this problem. For those experiencing difficulties, we also suggest ways to produce high quality slides.

Staining patterns are the result of everything that has been done to the specimen throughout its entire surgical and laboratory history. Factors that most often are identified as causing undesirable nuclear staining are:

Surgical procedures. Laser and electro-cautery techniques denature macromolecules and produce heat artifact. Heat artifact is generally marked by dark basophilic staining in nuclei and cytoplasm. Swabbing, washing or otherwise treating the surgical site before removal of the specimen also can seriously alter staining patterns. This is particularly evident if the affected cells are non-keratinized epithelium. Most often, surface cells stain lightly; cells may be swollen if acetic acid has been used.

Post-operative events occurring before fixation. The fate of the specimen immediately after removal from the patient may determine staining quality in unexpected ways. Ideally, specimens should be placed directly into an effective fixative. In many cases, this simply does not happen. Fixatives may be banned from the operating room. The surgeon or pathologist may wish to examine the specimen in the fresh state. Perhaps fixation is delayed as a matter of convenience or necessity to the surgical team, or the pathologist wishes to receive specimens unfixed so that any of several fixatives may be used after initial examination. In any case the specimen may be placed for a variable period on a wet or dry surface or into saline, any of which can ultimately affect staining.

Cells die almost immediately after removal from the body, and autolysis begins soon thereafter. Saline does nothing to prevent this damage. Placing fresh tissue on dry gauze or toweling will instantly dry out the surface cells, making them resistant to penetration by the fixative. In either case, autolysis is characterized by lightly stained nuclei lacking sharp chromatin detail. Curiously, biopsies frequently show these problems to a greater extent than larger tissues, probably because biopsies consist of a rich population of metabolically active cells.

Fixation. This is the most important factor governing staining. Zinc formaldehyde solutions offer the best all around fixation, providing a system whereby all specimens can be fixed in the same solution regardless of the staining protocol desired or the type of tissue. Zinc's effect on immunohistochemical reactivity is nothing short of astounding.

Alcoholic formalin, with or without zinc, can be used on the processor to provide rapid, uniform fixation. Fatty tissues and specimens which arrive late in the day are particularly improved with this treatment. Please refer to our catalog for a summary of cost-effective fixatives designed to improve the quality of fixation and staining.

Tissue processing and slide preparation. Improper processing can further denature macromolecules, almost always in ways that can produce unfavorable staining. Excessive exposure to alcohol, xylene and heat produce dark, unpatterned nuclei. Many labs would benefit from altering their processing schedules. Closed processors rarely need more than 45 minutes per station, less with small animal tissues. Heat should not be used unless extremely short processing times are required. Switching to an alternative dehydrant and xylene substitute often allows far greater control over processing, especially when a wide variety of tissue types are run together. We offer the most gentle products on the market: Pro-Soft Dehydrant and Pro-Par Clearant, both of which can improve staining problems by avoiding harmful denaturization.

Exposure to hot wax should be kept to a minimum, generally no more than a total of two hours on the processor. Remember that specimens held in the embedding center continue to suffer from the harmful effects of the heat. Embedding must be done quickly to avoid variability in the staining within a batch of specimens. Small and delicate specimens should be embedded first; leave large specimens and fat until the end. Color coded cassettes offer a convenient way to segregate these tissue types.

Occasionally, slide drying introduces heat artifact. We prefer to keep oven temperatures below 60°C to avoid dark smudgy nuclei and reduced immunoreactivity.

Staining procedures. If after all of this, the specimen remains in good condition, two final factors may exert their influence: the staining process and the selection of stains. Hand staining introduces batch to batch variability. Machine staining provides more uniform results, but often creates problems because the program must fit in to a finite number of stations. Linear stainers impose an additional constraint (time) that can make effective staining difficult if close attention is not given to the choice of reagents. Slide-to-slide variability on mechanical stainers is most often due to improper fluid height, unwise selection of reagents at strategic stations, or inadequate rinsing of tissue.

The choice and use of some of the non-stain reagents affect the outcome in subtle ways. Adequate deparaffinization is important in order to avoid pale staining. Three changes, three minutes each are recommended regardless of the type of clearant.Tap water before and after hematoxylin may or not be acceptable. Iron, sulfur and chlorine will produce weak nuclear staining. If your tap water has a noticeable odor or color, it is better to use deionized or distilled water. Seasonal variations, especially in chlorine content, present a particularly frustrating variable in staining. Highly alkaline (hard) water may be good for bluing, but it may create dark nuclear or background staining.

While nearly all hematoxylins can be used progressively, brief treatment in an appropriate acid rinse usually helps promote sharper contrast. The best acid solution is an acetic acid/alcohol mixture because it provides the greatest degree of control while protecting the integrity of the nuclear stain. Various bluing agents can produce differences in stain color or sharpness. The most critical consideration is to provide uniform bluing across the slide.

Eosin staining presents fewer staining problems, possibly because it is of lesser diagnostic significance. Good uniform counterstaining is best achieved by passing slides through an appropriate solvent, usually ethanol (never isopropanol), before entering the eosin solution. This has the added advantage of prolonging the life of the stain. Multihued effects can be obtained only with the properly fixed material, using a good eosin solution and following the stain with suitable alcohol rinses. Stain intensity is best controlled in those rinses. Never dilute alcoholic eosin with water to reduce potency.

Quality and types of stains. Finally, optimum staining requires the use of quality stains chosen for your specific purpose. Harris hematoxylin is the most widely used nuclear stain, probably because of its exquisite crispness. All good Harris solutions need to be filtered daily; those that do not simply lack the specificity of proper Harris hematoxylin. Stability of Harris is often questionable, and short shelf life is common with commercial products. We are proud of our Harris hematoxylin. Our unique manufacturing methods assure an extraordinary shelf life, the best in the industry. Unlike some Harris solutions, ours requires only a few minutes to achieve proper staining intensity. It is marked by an absence of background staining in nearly all applications. Our Harris Hematoxylin does not contain mercury.

Gill-type hematoxylins are favored by some because they stain goblet cells and they do not require filtration (other than to remove cellular debris). We offer two varieties: Hematoxylin-Normal and Hematoxylin-Extra. The former is used for routine staining; the latter is for frozen and plastic sections, or for creating a dark stain in a short time.

Our Eosin is a potent, selective counterstain that can be diluted with ethanol for those preferring paler colors. Either way, it produces beautifully differentiated tissue components.

Quality stains are hard to find, particularly in this age of corporate mergers and divisional spinoffs. Even harder to locate is technical backup for the stains. Our products are packaged with detailed instructions and suggestions so that you can tailor the stains to your preferences. Behind that is our toll-free technical service line. If you have problems or questions, give us a call. We probably have dealt with similar problems before and can provide rapid, competent answers. We can even provide processor and stainer programs for any machine, using any reagents.

We want to do more than simply sell you stains; we want your stains to be the envy of your professional community. We've done it for many others. May we assist you?