**4. Staining**

added to the water bath. The use of adhesives in the water bath promotes bacterial and fungal growth. Daily cleaning of the water bath with sodium hypochlorite solution (e.g., Clorox soap) is necessary to prevent contamination. Alternatively, a thin coat of albumin can be applied directly to the slide by dipping it into the solution or using your fifth finger (i.e., most ulnar and smallest finger). This latter process is referred to as "subbing." A newer idea is to use "plus" (+) slides. Treatment of the slide with a reactive silicon or polylysine compound chemically changes the glass, such that it bears abundant amino groups, which ionize to provide a positively charged surface. Sections which contain a preponderance of anionic groups, such as carboxyls and sulfate-esters adhere strongly to this modified glass. When creating a ribbon (what is a ribbon), i.e., a series of adjacent tissue sections, the hand wheel should be turned at a slow and even speed. Rotating the wheel too rapidly will cause sections of unequal thickness. The floatation bath should be heated to a few degrees below the melting point of the paraffin wax. Tap, deionized, or distilled water can be used. The ribbon should be gradually lowered onto the flotation bath to eliminate wrinkles and entrapped air. Air bubbles may be removed with a camel's hair brush or by submerging a slide under the ribbon. If the sections are wrinkling, a 70% alcohol solution can be added to the water bath prior to section collection. If necessary, sections may be separated, depending on their sizes, and each can be placed on a clean, pre-marked glass slide. Individual sections or tissue ribbons may be picked up by submerging a clean glass slide into the water bath at a ~45° angle, directly beneath the location of the section or ribbon. The slide should be lifted out of the water slowly to ensure that the sections lay on the slide. The slides should be drained vertically on a paper towel for several minutes before placing them onto a warming table (37–40°C). The slides should remain on the warming table, overnight, for 20–30 minutes at approximately 58°C or a few degrees below the melting point of the paraffin wax. Failure to drain the slides will create air bubbles under the tissue and decrease the section's adhesion to the slide. Air bubbles produce section unevenness and staining artifacts, making the final preparation difficult to examine with the microscope. Once the desired sections have been cut, the block can be removed from the block holder and sealed with molten paraffin wax to ensure that the tissue will not dry out and become brittle (blocks can last for weeks, months, or years) [7–10, 16, 17, 21, 22, 24–28].

12 Histology

**3.5. Problems encountered with sectioning tissue blocks**

Histologists are confronted often with difficult tissue blocks that will not section easily. This may be the result of, for example, brittle or shrunken tissue, improperly infiltrated tissues, or sections with, for example, holes or scratches in them. If the tissue block appears to be brittle, a 10% diluted ammonium hydroxide solution may be applied (via soaking) to soften the tissue to prevent cracking and to more easily facilitate sectioning. If sections have holes in them, this can be indicative of incompletely infiltrated tissue. This may be alleviated by placing the tissue block back in the heated wax bath to melt it and then proceed to re-embed the block. If artifactual scratches or tears occur across the tissue sections, this may be indicative of flaws or dirt on the cutting edge of the knife and may be alleviated by repositioning or replacing the blade. Alternatively, other problems can occur if the tissue block appears to be too soft or too hard. If too soft, a remedy may be to place the block tissue side down on several sheets of Kimwipes or paper towel in the freezer (−15°C) or a refrigerator (0–4°C) (chilling times may vary), prior to sectioning. This technique will help to harden the wax so that it better matches the hardness Staining of tissue slides is carried out by reversing the embedding process in order to remove the paraffin wax from the tissue to allow water-soluble dyes to penetrate the sections. This process is referred to as "deparaffinization." The tissue slides must be exposed to a clearing agent and subsequently taken through a descending alcohol series to water (also referred to as "bringing your slides to water"). Choosing the appropriate dye for a particular tissue slide is related to its ability to color otherwise transparent tissue sections and various cellular components of the tissue. The term "routine staining" includes the hematoxylin and eosin (i.e., H and E) stain. This stain is used routinely as it provides the pathologist or researcher with a detailed view of the tissue, clearly staining, for example, the cytoplasm, nucleus, and organelles. The term "special stains" refers to a large number of staining techniques, other than H and E, that allow the visualization of particular tissue structures, elements, or microorganisms that cannot be identified with H and E staining [6–10, 17, 21, 22, 24–28, 31–35]. Examples include, Masson's trichrome (e.g., skin; identification of collagenous connective tissue), GMS silver stain (e.g., lung; identification of *Pneumocystis* or *Aspergillus* spp.), Periodic acid-Schiff (e.g., kidney; identification of high proportion of carbohydrates, such as glycogen, glycoproteins, and proteoglycans), Perl's Prussian blue iron (e.g., liver; identification of ferric (Fe3+) iron in tissue preparations or blood and bone marrow smears), Ziehl-Neelsen (acid-fast bacillus) (e.g., lung; identification of acid fast bacilli), Alcian blue (e.g., intestine; identification of acid mucopolysaccharide and acidic mucins), Alcian blue and PAS (intestine; combination of staining properties of both Alcian blue and Periodic acid-Schiff for identification of similar tissue components), Gomori trichrome (blue or green) (e.g., submucosa, identification of muscle fibers, collagen, and nuclei) [36].
