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Preparations, Stains and Artifacts

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Slide 1: Most mammalian tissues have no inherent color. Under a light microscope cells and tissues have color because they were stained with dyes during slide preparation. Slides #1-13 illustrate this point. You will note that after staining with different stains the same structures appear somewhat different. You are responsible for knowing the stains (and what they are used to demonstrate) which are listed in the course syllabus. Tissue or organ names in this Unit are given for the sake of completeness. You will learn the morphology of different tissues and organs as the course progresses.

Tissues and organs are routinely embeddded in paraffin or paraffin/plastic mixtures for routine light microscopic sectioning. Thinner sections can be made if the samples are prepared for electron microscopy and are embedded in plastic (such as methacrylates or Epon). These latter sections are usually better preserved. Several of the following slides illustrate the difference in appearance of this type of preparation.

This slide is of a small intestine stained with the most common light microscope stain called hematoxylin and eosin (H & E). Hematoxylin stains nucleic acids blue, (nuclei and cytoplasmic accumulations of ribosomes are mostly what pick up this stain). Eosin stains the cytoplasm of cells, and many extracellular components such as collagen, pink to orange depending on the type of eosin used. This is a typical section of paraffin embedded tissue. This type of section is typically 5-12 μm thick.  

Slide 2: In contrast to Slide 1, this image is from a small intestine that was fixed for electron microscopy, which generally yields better preservation than routine light microscopic fixatives such as formalin. Such specimens are also embedded in plastic. Paraffin sections generally are 5-12 μm thick while plastic embedded sections are usually from 0.5 μm to 1.5 μm thick. Thinner sections appear sharper (compare the detail with Slide 1). Some plastic embedded sections are stained with toluidine blue without another (counter) stain such as can be seen in this image. Since the section is much thinner, many components stain more lightly (look at the nuclei, for example; compare with Slide 1).  

Slide 3: This plastic embedded section (of cartilage on the right and adjacent connective tissue) is thicker than Slide 2 and so stains more deeply. Metachromasia is the property of some dyes like toluidine blue which change color when bound to macromolecules with many repeating subunits such as the carbohydrates of cartilage matrix (M). As you can see, the metachromatic color of toluidine blue is purple.  

Slide 4: PAS stains neutral carbohydrates pink to red and the hematoxylin here is used as a counterstain to demonstrate the nuclei. Some of the things that PAS is useful to demonstrate are mucus (in this image in one cell glands called goblet cells that are interspersed among the cells of the lining epithelium at the free surface) and the glycocalyx of some epithelial layers such as this one. Mouse duodenum, Periodic-acid shiff-hematoxylin (PAS-H).

Remember seeing the TEM of the glycocalyx of the small intestine in Cell Biology? If not, recheck the Atlas.  

Slide 5: Mallory stain can be 2 stains together or three (called Mallory Triple Stain). Its primary utility is its staining of collagen blue. In this high magnification image of a section of peripheral nerve, the connective tissue which covers the nerve and contains a large amount of collagen is on the left of the image. The nerve fibers,in longitudinal section, stained red are on the right.  

Slide 6: Masson, like Mallory, is a triple stain which is especially used to stain collagen (green). In this slide of the external ear canal there is an epithelium at the upper surface of the field, a plate of cartilage in the middle and glands (stained red-purple) at the bottom. The connective tissue (containing green-stained collagen) is seen holding everything together.  

Slide 7: Reticular fibers are a special type of collagen fibers. They are usually stained black by methods such as Gomori's or Foot-Hortega stains (which contain silver salts). In this image of kidney cortex stained for reticular fibers the tubular structures in the field each appear to be outlined in black. These are the stained reticular fibers that are around each structure. In H & E stained sections reticular fibers cannot be distinguished from other types of collagen.  

Slide 8: The most used stains are are Verhoff's stain (which contains a kind of hematoxylin), Weigert's stain and aldehyde fuchsin. Slides are often just labeled "elastic stain". Elastic fibers or fenestrated laminae appear black or purple with these stains. As you can see in this slide of an aorta section, the wall of a large artery contains an abundance of elastic fibers and laminae. Without a counterstain other details of the vessel are not clear.  

Slide 9: Some microscope slides that you examine will not be of sections. One type is smears of peripheral blood or bone marrow. These are stained with triple stains (often called Romanovsky-type stains) which often contain Azure A, methylene blue, and eosin. They are used because they allow the differentiation of the different types of developing and mature blood cells. Two of the common ones are Wright's stain and Giemsa stain.

This slide of white and red cells from a peripheral blood smear stained with Giemsa illustrates the appearance of an eosinophil (has red staining granules, a type of lysosome, in its cytoplasm) and a neutrophil (which has a different kind of lysosomal granules that are minimally stained, i.e., neutral to, Giemsa). The small, darkly stained fragments of cells are platelets. Giemsa staining can also be used with some whole mounts (see Slide 12).  

Slide 10: Metal impregnation is not a stain in the classical sense but it is used, especially for nerve tissues (different technics are used to demonstrate neurons and neuroglia). Samples are soaked in solutions of silver or gold salts which deposit a layer of metal on cell surfaces or on specialized structures within cells. In the image of a Golgi stained preparation of cerebral cortex (a), neuron surfaces are black. Impregnation techniques do not demonstrate all cells in a tissue. Their chief utility is in allowing one to see the shape and processes of neural cells in sections that are often quite thick.

In the Cajal stained preparation of spinal cord (b), neuronal intermediate filaments in the cell bodies (perikarya) and cell processes (axons/ or dendrites) are stained dark brown. This allows the visualization of the nuclei in negative image (i.e., unstained- white arrow, although the nucleolus often does pick up some stain). In the cross sections of mylelinated nerve fibers at the right edge of the image, nerve cell processes are seen as dark dots surrounded by white halos of unstained myelin. Compare this appearance with the images of myelin in Slide 11.  

Slide 11: Osmium tetroxide is used as a stain in both light and electron microscopy. It is also used as a fixative as well as a stain for electron microscopy. Osmium reacts with lipids to yield a black stain.

In Panel a of this image of teased (i.e., whole processes, not a section) nerve cell processes (axons and /or dendrites), the lipid-rich myelin sheaths surrounding the processes are blackened by the osmium. The areas between myelin segments can easily be seen (arrows; these regions are called nodes of Ranvier). Examine Slide 5 and see if you can locate some nodes of Ranvier in the longitudinally sectioned nerve.

Osmium can also be used to stain myelin in sections of myelinated nerve cell processes (Panel b; here in cross section). Note that the myelin "doughnuts" are of different thickness and diameters. The nerve cell processes are central and stained pale yellow.

Panel c is a low power electron micrograph of cross sections of two myelinated nerve cell processes (N) that were treated with osmium after primary fixation with glutaraldehyde. At this low power, the myelin on each appears as a dark ring. The detail provided by the electron microscope shows that the myelin is surrounded by the cytoplasm of the cell that made it (a Schwann cell). If you are examining this image with the Nerve unit, notice the external lamina that tells you for sure that you are in the peripheral nervous system.

Panel d is an H&E image of myelinated nerve in plastic embedded tissue. It is cut in cross-section and comparable to panel b but at a slightly lower magnification. Compare and see that in this preparation the myelin is light pink and the central nerve cell processes appear as dark pink dots in the center of the myelin sheaths.  

Slide 12: In addition to blood smears and teased preparations, whole mounts are not sections. Indeed, the "whole thing", in most cases, is an object such as this piece of mesentery, which was fixed, placed on a slide, dehydrated, and stained. These preparations are quite thick by light microscopy standards but they offer a wealth of detail, especially in the third dimension. The large lymphatic vessel in this field is thin enough to see through. The large white arrow points to the leaflets of a valve inside the vessel. Some small blood vessels (capillaries and venules) can be seen to be filled with red blood cells. The dark purple spots are mast cells and the dark structure running diagonally across the upper right hand corner of the field is a nerve. This type of preparation is difficult to photograph and much more information can be gained by examining such a slide in the microscope.  

Slide 13: Various basic dyes demonstrate the areas of concentrated RER lamellae with free polysomes between them (together they are called Nissl bodies) as dark staining patches in the cytoplasm of neurons. Two neurons with well stained Nissl bodies are indicated by arrows. The basic dyes stain RNA particularly well and thus the nucleoli of these cells are also stained if they are in the section. There is no counterstain in this slide. (Spinal cord.)  

Slide 14: Artifacts can be caused at many different steps during slide preparation. Slides 14-20 illustrate some of the more commonly seen artifacts.

This image illustrates a diffusion artifact. As fixative penetrated this liver (from one direction), it pushed the glycogen to one side of each cell until it was stopped by the cell membrane. PAS-H stains glycogen red and you can see that each cell has most of its deeply stained glycogen located predominantly on one side.  

Slide 15: Light microscopic slide preparation techniques often cause dramatic shrinkage of cells and tissues. Some shrinkage spaces (white areas, especially between the cells and the basement membrane) are easily recognized in this slide. (Kidney medulla H&E.)  

Slide 16: The broad white area across the center of this field as well as the white spaces between adjacent cells are shrinkage artifacts. (Pancreas, H&E.)  

Slide 17: Folds are almost always seen as darker strips or areas, and when observing them in the microscope the increased thickness can be discerned. (Human penis, cross section. H&E.)  

Slide 18: In this image locate the large, light cracks, the fold (towards the upper right), the two pieces of extraneous tissue fragments, and the one dye precipitate (very dark, towards the lower left). (Spleen, H&E.)  

Slide 19: Post-mortem degeneration or inadequate fixation. Artifacts of this type are not easy to recognize at first, but the organ (kidney) is composed mainly of epithelial cells arranged in tubules. The areas with no apparent organization and masses of cells with ragged edges are degeneration or poor fixation artifacts. Note that the artifact is not uniform. Some structures are much better preserved than others. (Kidney, H&E.)  

Slide 20: Knife scratches. Nicks or irregularities in the blade used to cut a section cause parallel lighter stripes or white scratches across a section. (Tonsil, H&E.)  

Slide 21: Since sections may cut through cells and tissues in many different ways, it is important that you begin to recognize oblique as well as longitudinal or cross sections. In this slide the left and right sides of the field show the same tissue. On the right the epithelium is sectioned perpendicular to the basement membrane and the simple columnar epithelium is easily recognized, whereas on the left the obliquely sectioned epithelium is impossible to classify based on its appearance. The take home message is: What you see depends on how you slice it!

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