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Laboratory 5B: Connective Tissue Proper

Objectives:

1. Differentiate microscopically between the different cell types of CT proper andknow the functions of each.

2. Identify the microscopic appearance of each of the major classes of CT proper.

3. Know and recognize the components of ECM within the different classes of CTproper.

4. Correlate between structure, site and function for each class of CT proper.

Light Microscopy:

Cells: Draw each cell type and relate its morphology to its specific function. Knowwhich cells are resident cells, and which are wandering cells.

Slide 1: Subcutaneous Tissue Spread, Ferritin Injected, Rat, H&E

Move to a thinner region of the spread. At 10X note the pink staining collagenfibers, and at 40X, the thin, black elasticfibers. (See previous lab.)

Move to a region where blood vessels are prominent, but the tissue is not toothick. Locate the following cells and observe at 40X:

MacrophagesFibroblastsMast cells (Find these cells on slide 3 first.)Plasma cells

You may also have:Neutrophils

Adipose cells

Macrophages are the cells with the blue granules in their cytoplasm. The

animals were treated with ferritin, which has been phagocytosed bymacrophages. In some slides the blue looks more bubbly than granular.Be sure to look at both kinds of staining.

Fibroblasts are spindle shaped cells with oval nuclei. They may be easierto find on slide 3 first. The cytoplasm is indistinct in many cells.

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Mast cells (find these cells on slide 3 first) are oval cells with centrallyplaced round nuclei. The have small distinct (at 40X) purplish granuleswhich fill their cytoplasm. They are fewer in number than macrophages,and tend to locate in groups near blood vessels. If you are not finding

them, move to another area with blood vessels.

Plasma cells have eccentrically placed nuclei with prominentheterochromatin (cartwheel nucleus). Their cytoplasm is more basophilic,with a pale region adjacent to the nucleus in most cells. What causes thisstaining pattern? Plasma cells may be difficult to find in these slides. Ifyou have trouble finding plasma cells, find them on slide 196 instead.

Neutrophils (you do notneed to find these cells) are small cells with pinkcytoplasm and multilobed nuclei. In these slides the nuclei appear donutor C shaped. If they are present in your slide, they are most likely very

prominent.

Adipose cells appear as large, transparent bubbles that you can see otherstructures through. They have a thin strip of cytoplasm containing thenucleus at their perimeters. These cells usually appear in patches

After finding these cells, find them again on two of your neighbors slides (youshould be locating each cell type on 3 different slides).

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Slide 3: Connective Tissue spread, Rat, Safranin O and Trypan BlueIn this slide safranin O stains mast cellgranules red. Look for brilliant red

cells at 4X to locate mast cells. Note that they are not distributed evenlythroughout the tissue spread, but tend to occur in bunches, along blood vessels.Recall this distribution when looking for mast cells in slide 1. (Images next page.)

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Slide 134: Ileum, H&ELocate columnar epithelium on one side of the tissue. This epithelium is

lining finger like projections of CT (villi), many of which are cut in cross section.Look for eosinophils with bright red granules in the CT of these projections.

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Slide 25: Synovial MembraneThe majority of this slide is composed of adipose tissue; the remainder is

dense irregular CT. At 10X and 40X, identify adipose cells and fibroblasts.

Slide 196: Mammary GlandAt 4X, find a region of glandular tissue composed of tubules lined by

simple cuboidal epithelium. At 40X observe the areas of CT immediatelysurrounding the tubules and locateplasma cells, lymphocytes (appear asspherical nuclei), mast cells, and fibroblasts. Note: If you cannot quicklylocatethe cells mentioned, please ask for assistance.

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Classify This CT Proper:Connective tissue proper is classified by the density and orientation of matrix

fibers. What characteristics does each classification of CT have and how do thesecharacteristics support its function?

Slide 73: AortaViewed previously. Loose areolar CT properis found adjacent to the

arterial wall. Note the distribution of collagen and elastic fibers. What fills theempty white spaces? What are its constituents?

Slide 178: Testis

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Dense irregular CT propermaking up the organ capsule. Note sparsedistribution of fibroblasts.

Slide 25: Synovial membraneViewed previously. Adipose tissue and dense irregular CT proper.

Note fibroblasts in the dense irregular CT proper and fibroblasts and adiposecells in the adipose tissue. (See picture above.)

Slide 5: TendonDense regular (fibrous or collagenous) CT proper. Note the

organization of collagen and distribution of fibroblasts. There may also beareolar tissue at the edges of the tendon, and dense irregular CT properat oneend.

Slide 7: Yellow Elastic TissueDense regular (elastic) CT proper. Note the organization of collagen

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and distribution of fibroblasts in both longitudinal and cross section. I will notexamine you on recognition of dense regular elastic vs dense regular fibrous CTproper. Identify both as dense regular CT proper. Observe both longitudinal andcross sections. In cross section note the small number on nuclei and theirlocation outside the pink staining collagen fibers.

Slide 152: LiverReticular tissue. Note distribution of fine reticular fibers, and lack of

collagen and elastic fibers throughout most of this tissue. Reticular fibers (and

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cells that secrete them) constitute the only CT support for the functional cells inareas supported by reticular tissue.

Some slides to practice on:Slide 104: Trachea & Esophagus

Adipose tissue surrounds the esophagus.

Slide 41: Skeletal muscle

Locate dense regular CT proper.

Slide 53: Sciatic nerveLocate adipose and dense irregular CT proper.

Slide 65, 69 or 67: SkinLocate areolar, dense irregular, and adipose CT proper.

Micrograph folderPlasma Cell:

Note the distribution of organelles and relate this to the appearance of the

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plasma cell in light microscopy, and to the function of plasma cells.

Texts:

Junqueira:Figures Chapters 5 and 6

BermanFigures: 1-16; Chapter 2; 10-3; 19-24,27; in Chapter 21 find

micrographs of CT proper