At low power locate the zone of loose connective tissue that lies between the layer of glands and the thick layer of dense, eosinophilic smooth muscle. Many small blood vessels lined with simple squamous epithelium (endothelium) are present in the connective tissue.

With higher power, select a good area of loose tissue and note:

Collagenous fibers are of various sizes, stained pink with eosin, are coursing in various directions, and are cut in various planes. They appear ragged in sections.

Fine elastic fibers are present but difficult to distinguish with this stain, also because sectioning will leave only fragments of fibers. Look for some. They will be fine, homogenous threads, poorly stained but somewhat refractile.

Fibroblasts are shrunken; this is typical in routinely prepared sections. Nuclei are clearly defined and stain darkly. Thin cell processes may be seen on some cells. Other cell types are rare.

Look in the zone of loose connective tissue in the part of the section just beneath the epithelial layer. Note that the fibers course in various directions. Some of the thinnest fibers seen here are probable reticular fibers.

Identify fibroblasts, numerous lymphocytes, and plasma cells.

Look in the small areas of more normal loose connective tissue, nearer the muscle and elsewhere; identify collagenous and elastic fibers.

This slide has groups of mixed glands with a special epithelium that need not be identified, but their ducts may be lined with simple columnar or stratified columnar epithelium.

This is abdominal skin from a 3-week old rat, fixed to preserve mast cell granules, stained with toluidine blue (no counter stain).

Mast cells are abundant. Find some that show the entire cell: oval cell body, cytoplasm filled with purple granules (some may appear vacuolated), and a central small rounded light blue nucleus. Note the large size of the mast cells.

The epithelial-lined structures extending in from the surface epithelium are developing hair follicles.

Trypan blue, a “vital” dye, is used to demonstrate phagocytic activity of macrophages. A 0.5% solution was injected intraperitoneally into young rats on alternate days for two weeks. Macrophages all over the body ingest the trypan blue particles.

Slide 100, Mesentery and mesenteric lymph nodes, azocarmine. All nuclei stain pinkish-red with azocarmine; other tissues take only a faint background stain.

Look in the connective tissue of the mesentery for macrophages of various sizes with inclusions of trypan blue in their cytoplasm.

The lymph nodes are dense masses of lymphocytes supported in a reticular connective tissue stroma. Look for macrophages in the less dense areas.

A thick layer of dense irregular connective tissue underlies the stratified epithelium. Prominent structures seen in this layer are hair follicles, sebaceous glands, and sweat glands. Also present are small blood vessels and scattered fat cells.

Study the connective tissue. Note:

The density of the connective tissue layer.

The thick bundles of collagenous fibers, seen in various planes of section - cross, oblique and longitudinal sections.

Look for thin, refractile elastic fibers. They can be seen with high power and careful focusing.

Identify fibroblasts. Nuclei are seen, little or no cytoplasm.

Review the structure of stratified squamous epithelium. The eosinophilic, “stringy material” in the uppermost part of the epithelium is the stratum corneum that is sloughing.

The thick wall of the hair follicles is a somewhat modified stratified squamous epithelium continuous with the surface epithelium.

Sebaceous glands, associated with the hair follicles, are composed mainly of rounded cells filled with oily secretion which dissolves in prepared sections, thus leaving vacuolated cells that stain very lightly. The nucleus is centrally placed.

Fat is found in the subcutaneous tissue, below the layer of dense irregular connective tissue. It is present largely as adipose tissue.

Scan to locate the adipose tissue.

Recognize fat cells as large, clear cells.

In the larger masses of cells, note connective tissue septa, which form lobules of adipose tissue.

With higher magnifications:

Look at the large, more or less rounded individual fat cells, empty, showing the cell membrane and perhaps some of the rim of cytoplasm. (Fat has been removed during preparation.)

Look for an eccentric nucleus within the fat cell. Every cell will not show one.

Nuclei of fibroblasts are present between fat cells. It is not always easy to distinguish these from fat cell nuclei.

Look at the interlobular septa. What type of connective tissue forms these septa?

Recognize blood vessels in the septa and smaller vessels among the fat cells.