Fixation on Histology

Issues with Tissues


Having taught histology for over 30 years, one comment that I routinely hear is, “Everything looks-alike, it’s all pink and blue. How can I figure out which tissue is which?” This is no surprise as the routine stain in histology is a combination of Hematoxylin and Eosin, H&E. Hematoxylin stains tissue components, like nuclei, blue and eosin stains tissue components, like cytoplasm and collagen, pink.

You may be new to histology or more experienced but, after you troubleshoot H&E staining of your specimens, do you feel confident in being able to identify the basic tissues and their subtypes without the use of special stains? Each tissue and subtype has unique features that allow you to distinguish between them and knowing these will help you read H&E stained slides. There are 4 basic tissue types; epithelium, connective, muscle and nervous and each of these has unique morphological features that coincide with its function. Epithelium is located at a surface and is cellular and can be simple or stratified and is the only tissue type that has polarity, meaning it has distinct apical and basal regions. Muscle and nervous tissue are both cellular but lack polarity and connective tissue is the least cellular but its extracellular matrix (ECM) is the key feature allowing for its function. Understanding the basic function of the tissues help you to appreciate the morphology.

Epithelial tissue forms sheets of cells that cover the outer surface of the body and lines body cavities and hollow organs. Its many functions include protection, secretion, absorption, filtration and more. Epithelial tissue also forms endocrine and exocrine glands. Connective tissue can be classified as dense or loose depending on the abundance of fibers. Areolar loose connective tissue, the most diverse and abundant, is always associated with epithelium and offers support, nutrients and defensive function. Other types of connective tissue connect, support, and/or protect depending on their ECM. Muscle tissue allows for movement. Skeletal and cardiac muscle both have striations, meaning they have visible bands, but that is where the similarities end. Skeletal muscle is composed of long fibers (cells) with many peripheral nuclei while cardiac muscle has shorter branched fibers with a central nucleus. The fibers are attached to each other by intercalated discs. Smooth muscle lacks striations and has fusiform fibers with a central nucleus. Nervous tissue is very cellular but does not form sheets like epithelium, and is composed of 2 basic cell types, neurons and supporting cells. The neurons, nerve cells, commonly have processes (axon and dendrites) and form synapsis with other neurons or structures. The more abundant supporting cells are smaller and function in defense, myelin formation, nutrition and more.

In addition to the basic features, one must consider the plane of section to determine what tissue type is present. Organs and tissues are three dimensional and the problem with sections is that the specimen is now viewed as a two dimensional image. Orientation during embedding allows you to create a cross or longitudinal specimen. The difficulty is mentally going from a 2D section to a 3D specimen. Therefore, one must be able to distinguish tissues in different planes. For example, the characteristic striations of skeletal muscle are seen in longitudinal not cross section. The unique intercalated discs of cardiac muscle are not seen in cross section but knowing how the fibers of these 2 muscle types are arranged will allow you to identify the tissue with confidence.

There are numerous histology “look-alikes” with H&E staining. Three types of tissue that are easy to confuse are smooth muscle, dense regular connective tissue, and nerve in longitudinal section. Let’s try to identify the tissue below.

So, what are we looking at? Is the eosinophilic material the cytoplasm of smooth muscle or the collagen of dense regular connective tissue? The nuclei, are they fibroblasts or muscle cells? So is the tissue smooth muscle or connective tissue? Why isn’t it nervous tissue? Recall that muscle tissue is very cellular while connective tissue has fewer cells and an abundance of fibers. Nerve will appear bubbly or light as the myelin is removed in processing. At first glance looking at the ratio of hematoxylin staining nuclei to the eosinophilic staining material the ratio of blue to pink is about 1:3. The nuclei are closely packed in this specimen the correct answer is smooth muscle.

Tissue “look-alikes” can also be found within one type of basic tissue. For example, can you identify the different simple and stratified types of epithelium? To identify a surfacing epithelium, recall that it is located between 2 different environments and demonstrates polarity. To classify epithelium one considers the number of cell layers. If there is one layer of cells it is simple and if there are more than one layers of cells it is stratified. The shape of the cells in the apical layer further classify the epithelium. If the cells are flat it is squamous, if the height and width are the same like a cube it is cuboidal, and if the cells are taller than they are wide it is columnar. Histology students often confuse transitional epithelium with stratified squamous non-keratinized epithelium. This is understandable as both are types of stratified epithelia. Which type of epithelium is presented below?

The epithelium is the tissue located in the upper portion of the micrograph. Note that it is very cellular with many nuclei closely packed together. A noncellular basal lamina separates the epithelium from the underlying connective tissue. The tip of the black arrow indicates the basal region of the epithelium. Notice how the eosin stains the cytoplasm of the epithelium versus the collagen fibers of the underlying connective tissue. Many layers of nuclei are visible in this section and the apical layer(s) have flatten nuclei and are squamous. This is stratified squamous nonkeratinized epithelium. Areolar loose connective tissue is deep to the epithelium as expected.

Because of the resolution of the light microscope, it is not possible to see the plasmalemma (cell membrane) of the cells which is visible with transmission electron microscopy. To get an approximation of the cell size and shape one must look at the nuclear shape. In the stratified epithelium above, note that the nuclei in the basal layers are round and as they approach the apical region the nuclei are flat indicating a squamous epithelium. Nuclear morphology is also very important in identifying different types of cells in connective tissue. If you see a round nucleus, is it really round or is it the plane of section? Could it be an oval nucleus as in the fibroblast that is in cross section or is it the round nucleus of a plasma cell or a lymphocyte.

If these are some of the issues you have with tissues, you might want to attend my workshop, entitled “Issues with Tissues” (WS-16) on Wednesday, September 15, 2021 1-2pm at the NSH Convention.

Written by Amy Lynn Aulthouse, Ph.D., Professor, Biological and Allied Health Sciences, Ohio Northern University, Ada Ohio
Micrographs by A.L. Aulthouse

1 comment



08-18-2021 06:32

Thank you for your contribution Dr. Amy Lynn Aulthouse. It is a very good input as H&E is a universal and routine. Your article is very important especially in Africa where no or few options to other types of stain and depends upon H&E for diagnosis. See you on September 15, 2021.
Giorgis Yeabyo, ASCP, HTL
NSH Africa Representative
Addis Ababa, Ethiopia