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Unit 2: Connective Tissue
Connective Tissue Proper,
Adipose and Reticular Tissue
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Connective Tissue
Adult connective tissue contains a large amount of intercellular substance (fibers and ground
substances) and relatively few cells. Unlike epithelial tissue, connective tissue has relatively
little intimate contact between the cells that make up part of its structure. There are two main
sub-classifications of connective tissue. They are 1) connective tissue proper and 2) specialized
connective tissue. Connective tissue proper is subdivided further into loose (or sometimes
referred to as areolar) and dense. Loose (areolar) is ubiquitously distributed throughout the body
filling in areas between blood vessels and the substance of organs through which vessels course
and beneath the covering of the skin and lining of the GI tract. Dense connective tissue makes
up the structure of deep fascia, aponeuroses, tendons, ligaments, and the capsules of many
organs. Specialized connective tissue is further subdivided into adipose, cartilage, bone, and
blood. In this unit you will learn about connective tissue proper and two specialized connective
tissues, adipose and reticular tissues.
After completing this unit, you should be able to identify and distinguish between the following:
1. loose connective tissue
2. dense connective tissue
3. dense irregular connective tissue
4. dense regular connective tissue
5. adipose tissue
6. reticular tissue
4. elastic fibers
5. reticular fibers
6. collagen fibers
7. mast cells
8. macrophages
9. plasma cells
Also, after completing this unit in conjunction with reading a chapter on connective tissue in a
histology textbook and/or hearing a lecture on connective tissue, you should understand the
following:
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1. the importance of all three types of fibers
2. the importance of ground substance
3. the function of different connective tissues
There are nine specimens available for study of connective tissue. The specimens available for
study focus on the contrast between mesenchymal (embryonic) connective tissue and adult
collagenous dense regular and dense irregular connective tissue. One specimen is stained for
elastic tissue that demonstrates the difference between collagen and elastic fibers. Throughout
the specimens the principle cell of connective tissue, the fibroblast, is labeled. One of the
specimens is stained specifically for mast cells. The fourth specimen in the list (Reticular Fibers)
– lymph node demonstrates reticular tissue which is a specialized connective tissue. Specimens
of mammary gland and the intestine will also be used to illustrate loose connective tissue and
cells like plasma and mast cells. Adipose tissue will be illustrated with specimens from other
sections in the program. The order of study will be first the embryonic connective tissue,
mesenchyme, and then the three types of fibers, followed by dense, loose, and adipose tissues.
Finally mast and plasma cells will be demonstrated.
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Mesenchyme (embryonic connective tissue)
In the general histology section look under connective tissue and choose:
Mesenchyme (Embryo) – Hematoxylin-Eosin
All adult connective tissue is preceded in the developing embryo and fetus by mesenchyme.
This primitive tissue is composed of cells, fibers and ground substance like adult connective
tissue. Mesenchyme, however, is more primitive looking and has functioning cells that have the
potential of differentiating into fibroblast, chondrocytes, osteoblasts and other cells found in
adult connective tissue. Another difference is that mesenchyme has very small fibers. The most
dominant diagnostic feature of mesenchyme is that it is highly cellular.
Begin your study by selecting the 5x image of this specimen and scan it with the labels turned
on. Observed the labeled mesenchyme, blood vessel and brain anlage (anlage means precursor
or beginning of). Observe the difference in the density of blue stained dots between the
mesenchyme and brain anlage. In mesenchyme the cells are fewer per unit area thus resulting in
a light stain. Next scan the 20x image and find one of the labeled blood vessels. The cells in the
vessel are nucleated primitive red blood cells (just before birth the nucleated cells are replaced
with erythrocytes without nuclei. What type of tissue is lining the blood vessel? All blood
vessels are lined with endothelium that is composed of squamous cells and is classified as simple
squamous epithelium. Now observe the mesenchyme. Its appearance is largely reflective of a
collection of nuclei in cells the cytoplasm of which is hard to recognize. Continue your study of
this specimen by scanning the 100x image. At this magnification you can observe the nucleus of
the mesenchymal cells and their slim cytoplasmic processes. The space between the cells would
be occupied by ground substance in the intact tissue, but, in this image the ground substance,
mostly water-soluble glycosaminoglycans, was washed out during the preparation of the tissue
for microscopy. Before you leave this specimen observe and read the text related to the labeled
endothelium and primitive erythrocytes.
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Dense Connective Tissue
First we will study dense irregular connective tissue that makes up part of the dermis of skin,
capsules of some organs, and aponeuroses. In the general histology section under connective
tissue choose:
Collagenous Fibers (Skin) – Hematoxylin-Eosin
Scan the 5x image and locate the labeled epidermis and dermis. Recall that epidermis is
composed of stratified squamous keratinizing epithelium. Look around the regions labeled
dermis and notice the pink stained structures. These are collagen fibers. If you look closely and
compare the region below the label with the region above the label you should note that the pink
structures are larger below the label. Below the label is dense connective tissue and above the
label is loose connective tissue. The main difference is in that the size and prominence of the
collagen fibers are greater in dense connective tissue. In this specimen the higher
magnifications illustrate more details of dense connective tissue. Loose connective tissue will be
illustrated in a different specimen. Now select and study the 20x image noting the labeled
fibroblasts and collagen fibers. All you can observe about the fibroblasts are their nuclei that are
pretty flat. The pink staining collagen fibers have this staining result with eosin because of the
net positive charge of the collagen molecule that reacts with the net negatively charged eosin
molecule. The two collagen fibers that are labeled are sectioned longitudinally. There are many
collagen fibers in this image that are sectioned across (transversely). What should you conclude
about the arrangement of the collagen fibers in this specimen? Are all of them oriented in one
direction? Are they oriented in different directions? The collagen fibers in this specimen of the
dermis of skin are oriented in different directions. This is dense irregular connective tissue.
Now scan the 100x image and find the collagen fiber immediately below which there is a labeled
fibroblast. Carefully study the fibroblast observing its oval shaped nucleus containing
heterochromatin and euchromatin. Note the very light blue stained cytoplasm outside the
nucleus. Fibroblasts in adult tissue have a low activity of synthesis to maintain a turnover of
collagen molecules. In this state of low synthesis there is only a small amount of rough
endoplasmic reticulum needed and thus the cytoplasmic area is small. Note that the labeled
collagen fiber just above this fibroblast is sectioned longitudinally. Compare its appearance with
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the other labeled collagen fiber. It is sectioned transversely. Now we will study dense regular
connective tissue where all the collagen fibers and fibroblasts are oriented in parallel.
Dense Regular Connective Tissue
In the general histology section look under connective tissue. Choose:
Regular Connective Tissue, longitudinal (Tendon)-Hematoxylin-Metanil Yellow
In this specimen the nucleus and cytoplasm of the fibroblasts are stained shades of blue and the
collagen fibers are stained yellow. We begin with this specimen because the collagen fibers and
fibroblast nuclei are more readily observed compared to the hematoxylin-eosin stained
specimens. You will be observing the dense regular connective tissue in tendon. In tendons the
fibroblasts are called tendon cells. Also, in tendon the collagen fibers are wrapped by less dense
collagenous tissue into bundles. Between the bundles there are channels or pathways that carry
blood vessels. These pathways containing blood vessels surrounded by loose connective tissue
are called ‘peritendineum internum’. The collagen bundles are small (called primary bundles)
and large (called secondary bundles). Now select and scan the 10x image observing the
orientation of the tendon cell (fibroblast) nuclei in a parallel fashion alongside the collagen fiber
bundles. Now select and scan the 100x image observing in more detail the tendon cell and
peritendineum internum.
At this point you should choose the transverse sectioned tendon to observe the appearance of
dense regular connective tissue in the other plane of section. In the general histology section
under connective tissue choose:
Regular Connective Tissue, transverse (Tendon) –Metanil Yellow
Scan all three images, 5x-20x-100x. Note the smaller (primary) and larger (secondary) bundles
of collagen fibers. Note how all collagen fibers are sectioned across (transversely), especially
evident in the 20x and 100x images. Now you may wish to examine the hematoxylin-eosin
stained tendons to compare.
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This concludes our study of dense connective tissue. Next we will study loose (areolar)
connective tissue.
Loose (areolar) Connective Tissue
Loose connective tissue is characterized by smaller interwoven collagen fibers embedded in
more ground substance than in dense connective tissue. The fibroblast is the main cell that
produces and maintains the ground substance and collagen fibers. Loose connective tissue is
distributed throughout the body filling in the potential space between ducts, gland cells and
blood vessels. It is also present beneath the lining epithelium of the gastrointestinal, respiratory,
urinary, and reproductive tracts and just beneath the epidermis in the superficial layer of the
dermis of skin. Skin has several appendages, hair, sweat glands, and the mammary gland. The
specimens of mammary gland in WEBMIC have good examples of loose connective tissue so we
will begin our study with one of these specimens. In the organ histology section under skin and
appendages choose:
Mammary Gland, inactive – Goldner
This specimen is stained with Goldner’s stain. This is a trichrome stain that stains collagen bluegreen, nuclei black-blue, and cytoplasm red-violet. This is a female mammary gland from a
human in a non-pregnant state. Scan the 5x image with the labels turned on and observe the
labeled lobule composed of mammary gland ducts surrounded by loose connective tissue. At
this magnification you can easily observe that the density of collagen is much less in the loose
connective tissue of the lobule compared to the dense connective tissue surrounding the lobule.
Also note the scattered somewhat rounded white spaces outside the lobule among the dense
connective tissue. These are fat cells (adipocytes) and we will study them later in this lesson.
Select and scan the 20x image and find the labels ‘tubuloalveolus’ and venule (lower left part of
the image). Compare the density of collagen and cells between areas surrounding the two
structures. Collagen is significantly denser around the venule at the perimeter of the lobule.
Note also that the density of cells (judged by nuclei per unit area) is greater in the loose
connective tissue surrounding the tubuloavleolus. Thus you now have observed the difference in
the histology of loose and dense irregular connective tissue. Choose next the 40x image finding
the labeled fibroblast (in loose connective tissue) and the collagenous fiber (in dense irregular
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connective tissue). Complete your study of loose connective tissue by examining a specimen of
female non-pregnant mammary gland stained with hematoxylin-eosin. In the section of organ
histology under skin and appendages choose:
Mammary Gland, inactive – Hematoxylin-Eosin
Begin with the 5x image. The contrast between the loose connective tissue the ducts and
tubuloavleoli in the lobules and the dense connective tissue between the lobules is not as striking
with the hematoxylin-eosin stain as it was with the trichrome stain. As you study the 20x and
40x images the difference becomes more apparent.
In summary, loose connective tissue has smaller collagen fibers and more cells embedded in the
ground substance. It staining always appears lighter due to the lesser size and amount of
collagen fibers. Next we are going to observe reticular and elastic fibers. First let’s study elastic
fibers followed by reticular and then reticular and adipose tissues. To study elastic fibers select
a specimen in the general histology section under connective tissue. Choose:
Elastic Fibers (Skin) – Elastin (Verhoeff’s) – Nuclear Red-Picric Acid
This is a specimen of thin skin. The stain used renders nuclei-red, cytoplasm-light yellow,
collagen fibers-intense yellow, and elastic fibers-brown/red. Begin with the 5x image noting the
distribution of collagen and elastic fibers. You should conclude that there are more collagen and
elastic fibers in the deeper layer of the dermis beginning and below the label ‘dermis connective
tissue’ by observing the yellow collagen and brown/red elastin. This becomes even more
obvious when you examine the 20x and 100x images. Elastic fibers are distributed throughout
the dermis of the skin and are responsible for the elastic property of skin. You can test the
elasticity of the skin by taking a portion of skin on the back of your hand between your thumb
and index finger (what you have between your finger and thumb are two layers of epidermis and
the dermis). Pull the skin away from the back of your hand and then let go. Observe how the
stretched skin quickly rebounds to take the shape of the contour of the back of your hand. In
older individuals this rebound lessens until, in old age, doing the same maneuver results in a tent
like shape on the back of the hand which only slowly resumes the shape of the back of the hand.
Aging and ultraviolet light from the sun degrade the protein elastin until its elastic properties are
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significantly reduced. Elastic fibers are difficult to demonstrate in routine hematoxylin-eosin
stained specimens.
Reticular Fibers and Reticular ‘Specialized’ Connective Tissue
The third type of fiber found in connective tissue is the reticular fiber (collagen and elastic fibers
are the other two types). Reticular fibers are very small, on the order of 1 micron in diameter.
They are made up of type III collagen (collagen fibers are made up of type I collagen). Reticular
fibers cannot be visualized in specimens stained with hematoxylin-eosin mainly because they are
so small and contain so little collagen protein. The collagen of each reticular fiber is coated with
glycosaminoglycans. This molecule reacts with PAS (periodic shift reaction) and silver. To
study reticular fibers in the general histology section under connective tissue choose:
Reticular Fibers (lymph node) – Silver Stain—Nuclear Red
In this specimen you will observe reticular fibers as narrow black lines that are oriented in many
different directions. This specimen is a lymph node. An extensive network of reticular fibers
maintains the form of a lymph node. The term reticular is derived from the term rete, which
means net. Thus, the construction of the framework of a lymph node is like a net. All the
reticular fibers are secreted and maintained by reticular cells, a variant of the fibroblast. The
main functional cell of a lymph node is the lymphocyte. The reticular fiber network holds the
lymphocytes like a net holds fish. In addition to the lymph node, other organs that have a
reticular fiber network are spleen, liver, and bone marrow. Begin your study of this specimen
by scanning the 5x image noting the numbers of reticular fibers, one of which is labeled. The
small round lighter stained objects that will be recognized more readily in the higher magnified
images represent the lymphocytes. Examine the 20x and 100x images. In the 100x image you
can clearly see individual reticular fibers and can recognize the outline of the circular pale
staining lymphocytes. Now it will be instructive to make a couple of measurements. First find
the labeled reticular fiber in the right upper region of the image. First measure the diameter of
the lymphocyte using the measurement tool. Your measurement should be pretty close to 5
microns, the diameter of most lymphocytes. Now measure the thickness of the labeled reticular
fiber. Your measurement should be on the order of 1 micron. The actual size in real life is less
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than one micron. In the stained specimen the adhering silver molecules binding to the
glycosaminoglycans adds to the thickness.
At this point you have now seen and learned about collagen, elastic, and reticular fibers. You
also have studied loose, dense, and one specialized (reticular) connective tissues. Now, before
we look at some of the other cells in connective tissue, we will study adipose tissue.
Adipocytes and Adipose ‘Specialized’ Connective Tissue
Begin by examining an adipocyte (fat cell). In the organ histology section under blood and
blood vessels choose:
Vein-Hematoxylin-Eosin
This is a vein embedded in mesentery in which there is a significant amount of adipose tissue. In
the 5x image scan around the vein until you find the area labeled ‘adipose tissue’. Note the light
staining. In routine preparations lipid present in adipocytes is lost through the action of alcohols
and other lipid solvents used to prepare the tissue. Therefore, there is nothing left to stain.
Adipose tissue is a collection of adipocytes bound together with a collagenous wrapping and
permeated by capillaries and other small blood vessels. To get a closer look at fat cells
(adipocytes), go to the organ section and under blood and blood vessels choose:
Bone Marrow-Hematoxylin-Eosin
Here among the blood forming cells of the bone marrow are large adipocytes. Measure the
diameter of the adipocyte that is labeled. Its diameter should measure on the order of 100
microns. In this specimen it is not possible to observe the thin rim of cytoplasm and a single
nucleus that belongs to each adipocyte. In actuality an adipocyte in the adult has one large lipid
droplet. This is the typical histological appearance of a white fat cell. Brown fat is much
different. In brown fat there are many small lipid droplets and a large amount of mitochondria.
Brown fat is found in the fetus and in hibernating animals. To see the thin rim of cytoplasm and
nucleus of an adipocyte you will need to choose a specimen under general histology, glandular
epithelia-Goldner. In this specimen select the 2nd 20x image and you should see a labeled
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adipose cell appear in the microscope. Now you can observe the thin rim of cytoplasm and a
flattened nucleus at about 7 o’clock along the rim of cytoplasm. The nucleus and thin rim of
cytoplasm resembles a signet ring.
Plasma Cells and Lymphocytes
In loose connective tissue two cells that are found in addition to fibroblasts are plasma cells and
lymphocytes. Plasma cells are derived from B-lymphocytes. Lymphocytes are of two types, T
and B. T lymphocytes constantly circulate through the vessels and loose connective tissue.
Plasma cells derived from B-lymphocytes take up residence in lymph nodes, bone marrow, and
loose connective tissue where they synthesize and secrete specific antibodies. To teach you how
to recognize plasma cells and lymphocytes in tissue sections we will examine loose connective
tissue in a variety of locations. Let’s begin by examining the loose connective tissue beneath the
epithelium of the gallbladder. In the organ histology section under the gastrointestinal tract
choose:
Gallbladder – hematoxylin-eosin
Scan the 5x image and note the long fingers of tissue covered with epithelium. The mucosa here
is composed of simple columnar epithelium and loose connective tissue. Examine the loose
connective tissue more closely in the 20x image. In this image the loose connective tissue is
labeled lamina propria because it is the first layer of tissue beneath the epithelium, (which you
should recognize as simple, columnar). Now scan the 40x image and look for the labeled
lymphocyte. Measure its diameter. You should get a diameter on the order of 5 microns.
Observe the darkly blue stained nucleus and a very small amount of blue stained cytoplasm.
Compare the shape of the nucleus in the lymphocyte to the shape of the nuclei in the two labeled
fibroblasts. Note that the fibroblast nuclei are oval. Note also that there is one lymphocyte
labeled that is located within the epithelium. This is a common occurrence for T lymphocytes.
To illustrate plasma cells we will use a specimen from the body of the stomach. Go to the organ
section and under gastrointestinal tract choose:
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Stomach-Corpus-Hematoxylin-Eosin
Scan the 5x image and find the mucosa. We will look in the lamina propria of the mucosa of the
body of the stomach to locate a plasma cell. Select the second 20x image and find the labeled
lamina propria. This is the loose connective tissue beneath and between the glandular epithelium
of the stomach mucosa. Select the first 40x image and locate the labeled plasma cell. Note that
the cell is oval shaped and the nucleus is located at one side of the cell (eccentric position).
Plasma cells are larger than lymphocytes. Measure the long dimension of the cell. The cell
measures almost 9 microns compared to around 5 microns for the average lymphocyte. Even the
narrow dimension (the width) measures 7 microns. Plasma cells have more cytoplasm visible
than lymphocytes as can be observed here. The other feature is that the chromatin of the nucleus
of a plasma cell is arranged like the face of a clock. To observe that select the third 40x image
and find the labeled plasma cell. Also, compare the plasma cell nucleus with the nucleus of the
lymphocyte also labeled in this field.
Mast Cells
Mast cells are present throughout loose connective tissue and are typically arranged along and
just outside of small blood vessels. Mast cells secrete histamine, heparin and other factors. They
are important in the uptake of fatty acids from the blood into fat cells. To study mast cells go to
the general histology section and under connective tissue choose:
Mast Cells (Mesenterium)-CresylViolet-Nuclear Red
Scan the 5x image observing the labeled mast cells and fibroblasts. You can readily observe that
mast cells have more cytoplasm visible and they stain darker than fibroblasts. This is because
the mast cell stores its products in secretion granules and releases the content of these granules
when stimulated. Fibroblasts do not store their product but continuously release ground
substance, collagen fibers, elastic fibers, or reticular fibers as they are being synthesized. Select
the 100x image and find the labeled mast cell. Now you can easily observe the granules stained
violet and the nucleus stained red.
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Other Connective Tissue Cells
There are three other types of cells present in loose connective tissue. They are macrophages,
neutrophils and eosinophils. Macrophages are large cells with more cytoplasm than lymphocytes
or plasma cells. They have no granules and function to ingest other cells and bacteria.
Neutrophils are granulated, they have a lobated nucleus and are rounded, larger than
lymphocytes. Eosinophils have a two lobed nucleus and intense red granules. Neutrophils
migrate from the blood to loose connective tissue when attracted by signals from bacteria.
Eosinophils migrate into loose connective tissue when attracted by antigen-antibody complexes
or they can also be attracted by parasites. Macrophages are derived from monocytes of the blood
and, along with neutrophils and eosinophils will be studied when we study blood cells.
This now concludes your study of connective tissue. You have learned that there are two main
types of connective tissue: dense and loose. You have learned that there are three types of fibers:
collagen, elastic, and reticular. You have learned that fibroblasts are responsible for making all
three types of fibers. You have also learned that loose connective tissue has other types of cells
other than fibroblasts, namely, mast cells, lymphocytes, and plasma cells. You also have learned
that certain cells migrate into loose connective tissue from the blood: monocytes, neutrophils and
eosinophils.
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Sample Questions:
1. First order identification of a cell/structure or classification of a tissue.
Classify the tissue.
A. loose connective tissue
B. dense regular connective tissue
C. dense irregular connective tissue
D. cardiac muscle tissue
E. smooth muscle tissue
2. Second order question. A structure/cell/tissue is indicated and its function is asked.
The cell indicated by the number 1 functions to:
A.
B.
C.
D.
E.
synthesize collagen
synthesize elastin
synthesize reticular fibers
it does all of the above
synthesizes only collagen and elastin
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Connective Tissue Section Labeled Structures
The tables below are arranged by sections listing the specimens in the connective tissue
collection with the labeled structures by magnification. In reviewing you will find it easy to find
a structure by consulting these tables.
Mesenchyme (Embryo)-Staining: Hematoxylin-Eosin
20X
Blood vessel
Brain anlage
Mesenchyme
80X
Mesenchyme
Blood vessel
Primitive epidermis
400X
Endothelium
Primitive erythrocyte
mitosis
Mesenchyme cell - nucleus
Mesenchyme cell process
Collagenous Fibers (Skin)-Staining: Hematoxylin-Eosin
20X
Epidermis - stratified Squamous epithelium
Dermis - connective tissue
80X
Fibroblast
Collagenous fiber
400X
Fibroblast
Collagenous fiber
Elastic Fibers (Skin)-Staining: Elastin (Verhoeff’s)-Nuclear Red-Picric Acid
20X
Epidermis
Dermis - connective tissue
Smooth muscle
Hair
80X
Epidermis - stratified Squamous epithelium
Dermis
Elastic fiber
Collagenous fiber
400X
Elastic fiber
Collagenous fiber
Fibroblast
Reticular Fibers (Lymph node)-Staining: Silver Stain-Nuclear Red
40X
Reticular fiber
Lymphatic nodule
Blood vessel
80X
Reticular fiber
400X
Reticular fiber
Lymphocyte
Regular Connective Tissue, longitudinal (Tendon)-Staining: Hematoxylin-Metanil Yellow
40X
Peritendineum internum
Tendon cell
Tendon fiber
Primary Bundle
160X
Tendon cell
Tendon fiber
Peritendineum internum
Regular Connective Tissue, longitudinal (Tendon)-Staining: Hematoxylin-Eosin
40X
Tendon fiber
Peritendineum internum
Tendon cell
Primary bundle
80X
Tendon fiber
Peritendineum internum
Tendon cell
160X
Tendon fiber
Tendon cell nucleus
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Regular Connective Tissue, transverse (Tendon)-Staining: Hematoxylin-Metanil Yellow
20X
Secondary bundle
Primary bundle
80X
Tendon fiber
Tendon cell
Peritendineum internum
400X
Tendon cell
Tendon fiber
Regular Connective Tissue, transverse (Tendon)-Staining: Hematoxylin-Eosin
20X
Peritendineum internum
Primary bundle
Secondary bundle
80X
Primary bundle
Peritendineum internum
Fibroblast nucleus
Tendon cell nucleus
Mast Cells (Mesenterium)-Staining: Cresyl Violet-Nuclear Red
160X
Mast Cell
Fibroblast
320X
Mast Cell
Fibroblast
400X
Mast cell
Nucleus
Mast cell granules
Fibroblast
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