CONNECTIVE TISSUE
Two laboratory
periods are devoted to connective tissue:
 | The first laboratory examines the extracellular fibers of connective tissues. | |
| The second laboratory period examines the cells of connective tissues. | |
|
Blue Histology |
Prologue
This chart shows that the broad heading of "connective tissues" includes a variety of forms, some even mineralized. All have several properties in common, especially related extracellular constituents, and many contain the same or related cell types. The purpose of these next two laboratory sessions is to consider the most generalized form called "ordinary" connective tissues. We will deal with various specialized forms later.
"Ordinary" connective tissues are subdivided into loose (areolar) and dense varieties. However, there is no sharp demarcation between them. At one extreme, there can be no question but that one is dealing with a loose tissue. For example, the layer of "hypodermis" underlying the skin on the backs of your hands that permits the skin to slip around is very loose indeed. At the other extreme, the dermis of palmar surfaces is unquestionably very dense. Instead of having a hypodermal layer of loose tissue, the thick and tough dermis here is fastened directly to the underlying bone by way of a dense periosteal layer of connective tissue. Such differences in physical characteristics of connective tissue mainly reflect differences in the amounts and arrangements of the extracellular components, and one can find a continuum of variation as one moves around in the body. In some cases there is legitimate ambiguity in whether to call a tissue loose or dense.
Although ordinary connective tissue is classified mainly by structural criteria, it has at least two other sorts of functions. First, it provides a pathway for all nerves and blood vessels. Thus, capillary beds are in connective tissue spaces, and metabolic exchange is through the connective tissue compartment. Secondly, the connective tissues provide the substratum for very important defense mechanisms of the body. It permits the mobilization of cells capable of producing antibodies and other immune-type reactions, as well as for the cells we speak of as phagocytes or macrophages which are the scavengers of the body. Thus, connective tissues are crucial to all aspects of wound healing.
It is necessary to pay attention not only to the structural, extracellular components of connective tissue, but also to the various cell types. One generalization is that all of the cells of connective tissues derive from embryonic mesenchyme; thus, they are part of an extended family of cells with at least some common characteristics. Although some mesenchyme and derivative connective tissue cells spend all of their lives in their one original site; others, particularly those derived from blood-forming (hemopoietic) connective tissues, migrate about freely in the bloodstream to invade other connective tissue sites often far removed from where they started.
Connective tissues are a crucial and subtle part of histology. We shall devote two laboratories to ordinary connective tissue, first to its structural elements and then to its cells. Later laboratories will consider individual specialized connective tissues, bone, bone marrow, cartilage, lymphatic tissue and lymphatic organs.
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I. Extracellular components of Connective tissues
Objectives
You should become able to identify the following in your slide set:
| loose connective tissue | collagen fibers | |
| dense, irregular connective tissue | reticular fibers | |
| dense, regular connective tissue | elastic fibers (when stained) | |
| reticular connective tissue | elastic lamina (when stained) |
Slides
| D-5 | Tendon (H&E) | |
| D-24 | Carotid sheath (elastic stain) | |
| D-25 | Brachial artery & vein (Masson) | |
| D-26 | Subclavian artery (H&E) | |
| D-33 | Lymph node (reticulum stain) | |
| D-34 | Lymph node (H&E) | |
| D-129 | Sublingual gland (H&E) | |
| D-162 | Scalp (H&E) |
Optional slide
| D-7 | External ear (elastic stain, H&E) |
Slide descriptions
D-26, Subclavian artery
This slide demonstrates aspects of both loose and dense connective tissues after conventional staining with hematoxylin and eosin. The slide is labeled subclavian artery, that being the large circular structure easily visible with the naked eye. It also contains a variety of surrounding structures, all embedded in connective tissue. For example, the clusters of darker pink structures are sections of nerve bundles.
Although the inner part of the arterial wall is made up of smooth muscle, its outer half (its "adventitial" layer) is composed of dense and somewhat regularly arranged connective tissue (illustration). Examine it. One can immediately notice its sudden transition to very loose surrounding connective tissue with a fluffy appearance. In both areas the orange/pink-colored fibers that one sees are collagenous. Do not worry at this time about the appearance of smooth muscle, which makes up most of the inner wall of the artery. We will consider this tissue - in painful detail - later.
The major nerve bundles, cut transversely, also are wrapped in sheaths of dense connective tissue. Nerves need a substantial blood supply, so a plexus of relatively small blood vessels accompanies them. These include capillaries. Here, as usual, the blood vessels run in connective tissue (illustration). They truly are connective tissue components.
Examine the two large branches of the subclavian vein which have been split open and collapsed. Their walls are largely made up of dense connective tissue with a sudden transition to the loose type (illustration).
All of these structures are embedded in fairly loose connective tissue. In some regions substantial numbers of fat cells can be seen as large empty spaces (illustration). We will postpone their study for better preparations.
Turning to dense connective tissues, a good example of an irregularly arrangement is the dermis. If you feel your scalp you will realize that it does not have much give. It is a very tough layer resisting injury under many circumstances. For the present, look only at the dark red masses of collagen fibers in the dermis (illustration). Ignore the epidermis, hair follicles, sebaceous and sweat glands, nerves and other organized structures.
It is evident that the bundles of collagenous fibers can be massive in size. They run every which way. Some are cut transversely, some run in the plane of section for appreciable distance, but most are cut obliquely (that is histological-ese for "cut at a rakish angle") (illustration).
There are gaps between most of the collagen bundles. These have the fortunate effect of allowing you to discern the individual bundles easily but most of this empty space is artifact. It formed when the collagen bundles shrank during dehydration of the tissue. In life these gaps were small and filled with a (leached out) gel of ground substance. Therefore, use the size of the collagen bundles to distinguish dense from loose connective tissue instead of the spaces that may be present. In fact, loose connective tissue often shows less shrinkage artifact than does dense because the larger number of interlaced fibers forms a felt-work that shrinks as a whole instead of allowing individual bundles of fibers to pull away from each other individually to form internal gaps.
D-5 Tendon (H&E)
Connective tissues which transmit forces have collagen fibers aligned in parallel. These forms are called regular dense connective tissues. Tendons are prime examples. Note that nearly the entire mass of a tendon is composed of aligned, straight bundles of collagen (illustration). The fibroblasts that originally secreted these fibers are trapped into thin slits between the fibers. These cells are now totally inactive, as indicated by their greatly elongated, heterochromatic nuclei, and are properly called fibrocytes. Other dense connective tissues, such as ligaments, have less densely packed collagen fibers, but are still regular. In other places the fibers may run mostly in the same orientation but unless they are very highly aligned the tissue is considered to be irregular. Keep in mind, however, that a spectrum of forms extends from definitively regular dense connective tissue to completely irregular dense connective tissue.
Unfortunately you do not have a slide that easily shows a cross section of tendon. Some of your complex slides (e.g. of finger joints) do have areas of ligaments cut pretty much transversely. However, for now, content yourself with this picture from D-142 (illustration). It shows enormous straight bundles of collagen packed like a stack of uncooked spaghetti with a narrow band of other components (including the fibroblasts) between them.
D-24 Carotid sheath (elastic stain)
It is usually difficult to visualize the relatively small population of elastic fibers in the midst of the quantities of collagen that dominate ordinary connective tissues. However, special stains can be used to demonstrate their presence. Slide D-24 is just such a preparation, with elastica stained black.
Once again we see a fairly large artery, the carotid this time, vein and nerve bundle. The arterial wall has a great number of elastic lamellae stained black (illustration). They give this artery its notable elasticity. A moment's consideration should convince you that these wavy black lines have be sheets of elastica instead of fibers. Wavy fibers would not run in the plane of section for any distance at all.
Notice also that even the loosest connective tissue has a scattering of black fibers running every which way. Most are cut crosswise and therefore look like black dots (illustration). This is also the reason that the collagen (red) looks crumbly.
The sheaths of nerves, also present in this preparation, virtually are without elastic fibers, as is the adipose tissue found on this slide. Do not worry about the lymph node and other structures on the slide at this point.
D-25 Brach. artery& vein (Masson)
It is sometimes difficult to distinguish collagenous fibers from smooth muscle, or even nerve bundles. In such cases once again, a specific stain can come to our rescue by differentiating collagen from protoplasmic proteins. Slide D-25 is such a preparation, showing another major artery and its surrounding connective tissue. This slide is labeled as being stained with the "Masson" technique, but there are a number of variants, which we often lump together as "trichrome", or "polychrome" stains. They stain collagen blue or green in contrast to protoplasmic structures, which show up red or orange (illustration). Thus, for example, it is easy to estimate how much of the arterial wall is made up of smooth muscle (red) and how much is collagen (green). The same is true for veins and nerve bundles. Are you impressed with how abundant and pervasive collagen is? It is by far the largest component of most connective tissue Also, it is found only in connective tissues.
D-33, Lymph node: reticulum stain.
A variant type of loose connective tissue has for years been called "reticular connective tissue." It is characterized by very finely divided collagen fibrils, called reticular fibers. Histologists originally defined reticular fibers mainly on the basis of their affinity for "reticulum stains." This depends upon reactions which reduce silver and is now known to signify unusual quantities of carbohydrates associated with fine Type III collagenous fibrils. Reticular fibers can look kinky and they commonly give the illusion of branching (although electron microscopy has made it clear that this is not so).
Reticular fibers are abundant in almost all lymphatic tissue, as well as in bone marrow, where the mesh of finely divided fibrils supports a large population of cells, jammed together. Slide D-33 has one of these reticular tissues stained with a reticulum stain. The rich population of cells is only faintly seen because cells contain little carbohydrate. They are enmeshed in a three-dimensional network of the fibrils which do stain well. Look at this slide by eye to appreciate that the major variations in the architecture from one place to another are visible from the pattern of fibers. Do not worry at this point about the significance of these various parts, this is a task for a future laboratory on lymph nodes.
Now look at the reticular fibers at 400X power (illustration). You also can see a few seams of ordinary dense connective tissue running through the reticular tissue. Type I collagen contains a lower content of carbohydrate but enough to give it a brownish hue (illustration).
D-34 Lymph node (H&E)
You have another slide of a lymph node, D-34, which has been stained with H & E. It shows the dense masses of cells jammed together in lymphatic tissue, but the delicate reticular fibers are invisible. Take only a quick look at this slide for comparison: we will go over its complex structure in detail later. What you see are cells, cells, cells but no discernible reticular fibers. One lesson is that reticular fibers usually are impossible to see with light microscopy unless they have been specially stained (illustration). The other lesson, gained by simply holding the two slides up side by side, is that the connective tissue follows the same patterns as do the cells of tissues. No, this is backwards. In general, the connective tissues of organs establish the organs histological patterns and cause cells to develop or go where they should.
D-129 Sublingual gland (H&E)
Before concluding this exercise it is well to consider in a little more depth how connective tissues relate to associated epithelial components. The connective tissue forms the stroma (stromal compartment) of an organ. Its functions are supportive. The tissues that carry out the specific functions of an organ such as a gland are usually epithelial, and constitute the parenchyma. This distinction is one of the most fundamental principles of histological organization. The typical relationship is well shown in slide D-l29 of the sublingual gland (illustration). The functional glandular masses of highly cellular epithelial tissue are obvious. The stromal compartment surrounds this parenchyma and includes the vascular bed. Coarser seams of connective tissue support larger glandular subdivisions (lobules). These seams branch and invade the lobules to surround even the smallest units of parenchymal tissue (acini) with delicate sheets of loose connective tissue (illustration). Where there are major blood vessels and ducts, further support is provided by rather thick sheets of relatively dense connective tissue.
Stromal tissue is adaptive. It exists in the body in forms appropriate for the local metabolic activities. Its cell population is also adaptive to meet the defense requirements of particular situations.
Optional slide
D-7 External ear (elastic stain + H&E)
This is a section through the top of the external ear or pinna. It is supported by a sheet of elastic cartilage which you can feel on your own ear. Ignore it and the various other specialized structures embedded in the loose connective tissue for now. What interests us is the dermal layer of the skin. A substantial component of elastic fibers is incorporated into it. This section was treated to differentiate these fibers (with black coloring) from the pink of the collagen stained with eosin. Compare the delicate fibers in the loose connective tissue right underneath the stratified squamous keratinized epithelium with the coarse fibers of the dense connective tissue farther down (illustration). The bundles of collagen also reflect this difference.
Going back to the parting comment on an earlier slide, didn't I warn you that dense C.T. often shows more obvious (artifactual) spaces between fiber bundles than loose C.T. (illustration)? So base your classification on the thickness of the fibers and not the white spaces between them. Another important clue to the denseness of connective tissue is that looser forms tend to be more cellular. This difference shows up better with H&E than when the elastic fibers are heavily stained black, but you probably can still make it out here.
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II. Cells of connective tissue
Objectives:
You should become able to identify the following in your slide set:
| fibroblasts | |
| histiocytes (tissue macrophages) | |
| mast cells | |
| adipocytes | |
| adipose tissue (yellow fat) | |
| brown fat | |
| stroma | |
Slides
| D-2 | Adipose tissue (H&E) | |
| D-4 | Connective tissue spread (H&E, neutral red, trypan blue) | |
| D-26 | Subclavian artery (H&E) | |
| D-107 | Duodenum (H&E) | |
| D-131 | Adrenal gland, monkey (showing brown fat) | |
Optional slides
| D-3 | Umbilical cord (toluidine blue) | |
| D-143 | Fetal skull (H&E) |
Slide descriptions
D-4 Connective tissue spread (H&E, neutral red, trypan blue)
There is an alternative to tissue sectioning for studying loose connective tissue. This involves peeling back a piece of skin to get at the hypodermis in a living but narcotized animal. The tissue is made edematous by injecting a little saline solution to spread the fibers apart. Then small bits of the tissue are clipped out and teased on a glass slide to produce "whole mount spreads" instead of sections. The mounts are immediately fixed and stained. Slide D-4 is such a preparation. What one ends up with is considerably a matter of chance. It is obviously impossible to standardize this procedure, so share slides with your neighbors, if necessary.
Start first by identifying fiber types. The fibers are not cut but run for long distances until they get lost in a tangle of other fibers or tissues. Elastic fibers behave very much like rubber bands. They will be stretched into straight thin lines or recoiled into spirals where they have snapped. They can truly branch, and are sufficiently refractile to have an appearance distinctive from the collagen even with little or no stain. Bundles of collagen fibers look like pale pink ribbons of various widths (illustration).
In studying the cells in the spreads, be sure to choose areas where cells have been well preserved, and were not dried out or burst during the preoperative procedures. Also avoid areas which were not well spread, for one cannot see adequately through thick masses of tissue. This is important. You will be able to find the lightly stained nuclei of fibroblasts in quantity, although their cytoplasm will be so pale as to be generally invisible. These nuclei have the shape of oval platters. They will appear oval and pale if you look down on their flat face, but will resemble relatively dense cigars if looked at on edge. Fibroblasts represent perhaps 40% of the cell population in connective tissue of this sort (illustration). About an equal number of cells are histiocytes (= macrophages = phagocytes). These represent a separate population of cells which are derived from bone marrow. Resting macrophages look very much like fibroblasts. However, after they have consumed something, the cells often can be identified by their cytoplasmic contents without any special stains. If they have been consuming fatty material macrophages will appear vacuolated (the fat will have been extracted during processing), and the pathologist speaks of them as "foam cells." ( illustration from slide D-34, and illustration from D-99 as you will observe later). If they have been engulfing red blood cells, brown pigments accumulate in their cytoplasm that can be quite conspicuous (illustration from the spleen D-38, another slide for another day). In the lung, so called "alveolar macrophages" typically have ingested particles of dust and soot, again making them easy to pick out
One can play a trick with experimental animals to identify macrophages easily in any part of the body. This is to inject a colloid pigment such as trypan blue, carmine or India ink into the peritoneum of a living animal. After a week of injections the animal is sacrificed. Phagocytes all over the body will have picked up the colored material. The rat which contributed its tissue to slide D-4 received trypan blue, and the population of histiocytes in the tissue spreads is beautifully marked by the ingested purple particles (illustration). The slide has also been lightly stained with H & E to reveal the other cells and extracellular components. Only the histiocytes have phagocytized the trypan blue to any appreciable extent so that this procedure demonstrates a clear-cut functional difference between the macrophage system and any other population of cells.
A third cell type to look for in the slide D-4 connective tissue spread is the mast cell. These cells are large and their cytoplasm is distinguished by large numbers of small granules containing heparin and histamine (among other secretory products). The way our slides have been stained, with neutral red, these granules are a brilliant red. Mast cells tend to cluster along arterioles (illustration). Hence you should look for them in thicker areas of the tissue spread, where their granules will make the cells stand out at both 100X and 430X magnification. However, the numbers of mast cells vary greatly from one slide to another. It is even possible that you will not be able to find any without turning to your neighbor's slide (let me know if this is the case and I will try to find you another copy of the slide to look at).
The remaining cell types that you may encounter will be endothelial cells of capillaries or invaders from the bloodstream, particularly lymphocytes and eosinophils. These can be studied better elsewhere.
D-26 Subclavian artery (H&E)
You already turned to this slide to examine the fibers of loose and dense irregular connective tissue. It also shows the typical appearance of the cells in sections of ordinary connective tissue. At high magnification, look at some of the cells in the areas of loose connective tissue (illustration). Most are fibroblasts, characterized by elongate nuclei and wisps of cytoplasm extending beyond either end. Occasionally other cell types may be found, especially macrophages and white blood cells. In looking for macrophages you can appreciate how useful it was to have vitally stained this class of cells with trypan blue in slide D-4. Here it is almost impossible to tell the macrophages from the fibroblasts. White blood cells are rare in this particular tissue. The tissue came from a well-protected area of the body so that there had been no particular demand for protective white blood cell types to invade it.
D-2 Adipose tissue (H&E)
Fat cells (adipocytes ) are another important cell type in loose connective tissue. Isolated fat cells or small clusters are mixed in various proportions in many connective tissues. In protected places, essentially pure adipose tissue often can be found, filling crevices and niches. Slide D-2 is an example.
Some mesenchymal cells apparently differentiate before birth into preadipocytes which are programmed to become fat cells (adipocytes) at a later time. Given an adequate diet and hormone environment, they first start accumulating fat in numerous small droplets. Eventually, these coalesce into one big drop which pushes aside the nucleus and cytoplasm. Conventional techniques of slide preparation use organic solvents so that the fat droplet almost invariably is lost in histological preparations. One sees only a huge empty hole where the fat droplet was outlined with a narrow red band. This appearance has earned them the name of "signet-ring cells" . You see nuclei in only a small percentage of fat cells (illustration). This is because the nucleus occupies only a small region and most cells get sectioned in ways that miss that structure. A curious feature, which you cannot see here, is that each fat cell has its own basal lamina. Yes! Some connective tissue cells have basal lamina in addition to epithelia, whereas many do not (e.g. fibroblasts, macrophages and mast cells). Also, the cells in adipose tissue are supported mainly by reticular fibers, again invisible to the light microscope without special staining.
Fat cells constitute a metabolic reserve for the body. Starvation can lead to a real loss of fat and an apparent loss of fat cells. However, the depleted fat cells have only reverted to the appearance of fibroblasts. The propensity of these committed cells to reacquire fat persists, drat them!
Note that adipose tissue is richly supplied with blood vessels but wait until a future laboratory before trying to distinguish capillaries, small arterioles and venules from one another. The important point at this time is that adipocytes and adipose tissue are metabolically active and not just inert storage depots.
Fat tissue is not always well preserved on histological slides. It is easy to allow the delicate rims of cytoplasm of fat cells to partially collapse after the fat has been extracted. If you return to slide D-26 you can see the way fat cells often look in routine sections (illustration), ugh.
D-131 Adrenal gland, monkey (showing brown fat)
Besides ordinary "unilocular" fat cells seen above there is another variety called brown fat. It gets its name from the color of fresh tissue due to its rich content of mitochondria. Instead of having a single droplet of lipid, brown fat cells are multilocular. The stained cells have a sponge-like appearance with many small holes separated by partitions of cytoplasm. A collection of tiny fat droplets has much more surface area than the single droplet of ordinary fat cells. This allows a much higher rate of metabolism. The main function of brown fat tissue is to produce heat and this tissue is especially prominent in animals that hibernate. Adult humans do not have brown fat but it does occur in babies, around the shoulders and back. (The example here is from an adult monkey.)
D-107 Duodenum (H&E)
In well-protected places, ordinary connective tissues contain modest populations of cells, mainly fibroblasts, endothelial cells of blood vessels, and perhaps fat cells. However, underneath vulnerable wet epithelia other types of cells are necessary to protect the tissue from invading pathogens. Most of these cells migrate in from the blood. Such a connective tissue can be seen here in the duodenum. You have already examined the columnar epithelium over the villi of this slide. Now turn to the connective tissue directly underneath that epithelium. Note how cellular it is (illustration). Lymphocytes are the predominant immigrant cells, but there are also eosinophils, with their bright orange granules, macrophages and plasma cells, with their "clock face" or "cartwheel" nuclei. Do not worry about trying to sort out the cell types. This will be a task of a future lab period. For now, just note the abundance and diversity of cells in this connective tissue. Highly cellular loose connective tissues, such as seen here, often are supported by delicate reticular fibers in addition to coarse type I collagen fibers as discussed earlier. Note that the cellular connective tissue directly under the epithelium (the lamina propria) abruptly changes to ordinary loose connective tissue at its boundary with the underlying submucosal layer (illustration).
This change emphasizes the fact that the connective tissue right under an epithelium is almost always visibly specialized to support the overlying epithelium. Sometimes it is the vasculature or looseness of the tissue or the presence of glands which highlights the distinctiveness of the lamina propria. In this case it is the abundant protective cell population. The lamina propria is more closely related functionally to the overlying epithelium than the underlying connective tissue. Hence, we histologists refer to the lamina propria together with its epithelium as a mucosa (= mucous membrane) and designate the underlying tissue as a submucosa. (But, as they say on cheap tickets to Sydney, "restrictions apply". To be a mucosa the epithelium must be "wet", epidermis will not do. Also the epithelium must be connected to the outside, such as the case here with the lumen of the duodenum continuous with that of the stomach, esophagus, pharynx mouth and outside. The epithelium that lines a closed body cavity such as the pleural cavity around the lungs is part of a serosa instead of mucosa.) Most mucosae contain goblet cells and mucous glands. Their secretions drain to the outside, carrying bacteria, debris, etc with them. Serosae are wet but their lining epithelium (simple squamous) normally absorbs as much fluid as it secretes.
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Optional slides
Almost every slide in your set has ordinary connective
tissue in it, especially as stroma of various organs. The two optimal slides suggested
here show fetal connective tissues. Neither slide is particularly easy to examine
and you may feel content with just looking at the computer images.
D-143 Fetal skull (H&E)
Mesenchyme or "fetal connective tissue" is the source of all connective tissues in the adult. Mesenchymal cells resemble fibroblasts but tend to have somewhat more cytoplasm. This resemblance belies the enormous difference in potential of mesenchymal cells and fibroblasts to differentiate into other cell types.
D-143 shows mesenchyme, as well as ordinary
connective tissue, embryonic bone, blood vessels, early stages of skeletal muscle
and so forth that are differentiating from it. Mesenchyme is characterized by
having only the most delicate occasional collagen fibers. Once it becomes definitive
ordinary connective tissue collagen fibers can be seen. Using this as your criterion,
find an area of tissue between the skin and the layer of bone spicules which is
still at the mesenchyme stage (illustration).
You may be able to see that the mesenchymal
cells have more cytoplasm than is visible on fibroblasts in the adult connective
tissues. If you look carefully you can also find other regions in which the mesenchymal
cells have turned into fibroblasts and begun laying down collagen fibers.
D-3 Umbilical cord (toluidine blue)
One special fetal organ that is especially easy to obtain is the umbilical cord. It is composed mainly of mesenchyme but of a type somewhat different from that in the other fetal organs. It has much more ground substance and substantially plumper cells. For years obstetricians have known this tissue as "Wharton's jelly," which indicates its consistency. The less imaginative histologists use the less imaginative name "mucoid connective tissue". The mesenchymal cells on D-3 are only slightly differentiated, except in the immediate vicinity of the three blood vessels visible to the naked eye. Scan this tissue at low power (illustration). Then go to 40X and choose a relatively darkly stained area away from the blood vessels. The mesenchymal cells have multiple plump arms of cytoplasm which are attached to one another to form a network with spaces slotted in between (illustration). Any collagenous fibers are too fine to be seen distinctly, and the extracellular space is dominated by amorphous ground substance, rich in hyaluronic acid.
A special metachromatic stain has been used here to distinguish the cytoplasm in the arms of the mesenchymal cells from the surrounding amorphous ground substance. Toluidine blue is a purplish compound whose absorption spectrum changes upon complexing with highly ionized polysaccharides. Thus, cytoplasm appears pale blue, nuclei darker blue and ground substance, reddish. In many places, the ground substance has shriveled and clumped during the dehydration of the tissue, leaving holes with reddish tatters around them. These mesenchymal cells have much fleshier arms of cytoplasm than do fibroblasts. Unfortunately, toluidine blue slowly fades from exposure to light and your slides are pale compared to their rich color forty years ago.
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