Connective tissue observation experiment
Connective tissue observation experiment
1. Understand the characteristics and classification of connective tissue.
2. Know the fibers and various cellular components of loose connective tissue.
3. understand the basic structural features of dense connective tissue, adipose tissue, cartilage and bone.
4. understand the methods of making flat slices of fresh material of loose connective tissue and the methods of biopsy staining.
Operation method
Connective tissue observation experiment
Materials and Instruments
Rats Mice Move I. Materials and utensils For more product details, please visit Aladdin Scientific website.
Saline solution, glycerol, Ritter's stain.
Dissecting set Syringe and needle Dropper Slide Coverslip Microscope
Rats or mice. Flat slices of subcutaneous connective tissue stained by live staining and elastic fiber staining and H-E staining, trachea sections (H-E staining), small intestine sections (H-E staining), flat slices of mouse mesentery (Sudan III staining), Achilles tendon sections (H-E staining), lymph node sections (H-E stained), silver-stained sections of lymph nodes, and bone mill sections. Electron microscopic pictures of loose connective tissue.
1% trypan blue saline solution, glycerol, Wright's stain.
Dissecting set, syringe and needle, dropper, slides, coverslips, microscope.
II.OPERATIONS
Loose connective tissue
(I) Preparation and observation of live stained specimen of loose connective tissue
Take a rat (or mouse), inject a little 1% Tapan Blue saline solution from the skin, and kill the rat after 30 ~ 60 min. The skin was cut open and a small piece of subcutaneous connective tissue was cut near the injection site and placed on a slide. Then, holding a dissecting needle in the left and right hand, the tissue was picked apart and spread flat on the slide. After the specimen dries slightly, 1 to 2 drops of glycerol are added and the slide is covered for observation.
1. Observe with low magnification to distinguish collagen fibers and elastic fibers. Collagen fibers are not colored and are transparent white. Elastic fibers are thinner than collagen fibers and are light yellow.
2. High magnification observation Distinguish fibroblasts and macrophages according to the shape of the cells, the shape and staining of the nucleus, and the granules in the cytoplasm. Fibroblasts are flattened with many protrusions. Macrophages are irregular in shape and contain blue granules in the cytoplasm.
(B) Observation of subcutaneous connective tissue plain slices of cats or rabbits stained with Taipan blue in vivo and Weigert's elastic fiber stain and stained with H-E contrast.
(1) Low magnification observation: Select the thin and uniform specimen for observation, and observe the fibers crossing into a network and the connective tissue cells scattered between the fibers.
2. High magnification observation: first distinguish the fibers, and then distinguish the cells. This method can show collagen fibers and elastic fibers. There are connective tissue cells scattered between the fibers, and it is mainly required to distinguish fibroblasts and macrophages.
(1) Collagen fibers are stained pink in the form of thin bands of varying thickness. They are interspersed and numerous. Collagen fibers are indistinguishable. Sometimes the collagen fibers are wavy.
(2) Elastic fibers Dyed dark purple-brown, their broken ends are often curled. Thinner than collagen fibers, single, with branches, linked into a network.
(3) Fibroblasts are the most numerous, the cytoplasm is lightly stained, the outline is not very obvious, and it is flattened with many protrusions under close observation. The nucleus is large, mostly oval, stained blue-purple.
(4) Macrophages The cell shape varies, round, oval or irregular. Because of the live staining, it can be seen that its cytoplasm contains phagocytized Tepan blue particles. Cytoplasm staining is deeper, the cell outline is clearer than fibroblasts. The nucleus was smaller, round, oval or kidney-shaped, and the staining of the nucleus was also darker, dark blue-purple.
In addition, some granulocytic leukocytes and lymphocytes can be seen in the plain film.
(III) Observation of mast cells
Using mice (or rats) as materials, take a little subcutaneous connective tissue to make a flat sheet. After sufficiently drying, add Ritter's dye solution (Ritter's dye 0.1 g dissolved in 60 ml of methanol) drop by drop, so that the dye solution will completely submerge the material. After 1 min of dyeing, an equal amount of distilled water was added drop by drop and mixed well with the dye solution. After standing for 5 ~ 10 min, pour off the dye solution, wash with tap water, and leave it to dry before microscopic examination without sealing.
Mast cells were found in piles at the edges of the blood vessels in the flat piece with low magnification first, and then observed with high magnification. Mast cell cytosol is large, round or oval. The nucleus is round and not colored. The cytoplasm was filled with coarse reddish purple or bluish purple granules. Sometimes the degranulation of mast cells can also be seen.
(d) Observe the morphology and structure of plasma cells and loose connective tissue on paraffin sections with rabbit tracheal sections (H-E staining) and compare them with flat sections of loose connective tissue.
The loose connective tissues on the sections were not as well stained as the flat sections, where the fibers were mostly severed. The morphology of the loose connective tissue on sections will often be encountered in future studies of organ histology, so attention should be paid to it.
1. low magnification observation In the deeper layers of the epithelium, a slightly lighter stained structure is found, that is, the loose connective tissue.
2. High-magnification observation Mostly collagen fibers, stained pink, thicker. Mixed with irregularly distributed elastic fibers, but elastic fibers are not easy to distinguish on the section. Between the fibers are long oval nuclei stained blue-purple, mostly those of fibroblasts. In this area look for an ovoid cell, the cell's nucleus is located on one side of the cell, chromatin is arranged in a radial pattern, the cytoplasm near the nucleus has a lightly stained area, this cell is a plasma cell.
(e) Demonstrate the observation of the electron microscopic structure of loose connective tissue.
1. Scanning electron microscope structure with connective tissue scanning electron microscope picture observation, distinguish collagen fibers, fibroblasts and macrophages.
2. Transmission electron microscopy structures observed with connective tissue transmission electron microscopy pictures.
(1) Collagen fibers The longitudinal section of collagen fibers can be seen to have alternating light and dark periodic transverse striations, with a transverse striation period of about 64 nm.
(2) Fibroblasts The cells were polygonal. The cytoplasmic protrusions are long, and the endoplasmic reticulum and Golgi complex are well developed in the cytoplasm, and the nucleus is large.
(3) Macrophage Cell surface with microvilli, cytoplasm with a large number of lysosomes, near the edge of the cell with more vesicles. The nucleus has fine nucleoplasmic particles, which are mostly gathered on the inner surface of the nuclear membrane.
Dense connective tissue
Observed with longitudinal sections of frog Achilles tendon (H-E stain).
(A) Low magnification observation
Bundles of collagen fibers stained red can be seen in a parallel and close arrangement.
(B) High magnification observation
The collagen fiber bundles are thicker, and the bundles are composed of many collagen fibers arranged in parallel, but it is not easy to distinguish them on the section. Between the bundles of fibers are arranged in a single row of elongated tendon cells (fibroblasts), but in the section can only be seen stained blue-purple oval or rod-shaped nuclei. The nuclei of two neighboring cells are often close together. The cytoplasm is not easily visualized.
Adipose tissue
(i) Observation of sections of small intestine or trachea (H-E stain)
1. Observation with low magnification In the outermost layer of the loose connective tissue of the small intestine or trachea, dense groups of round or polygonal vacuoles, i.e., adipocytes, can be seen. Since the fat droplets in the cytoplasm were dissolved by alcohol and xylene during the preparation process, they were in the form of vacuoles. There is loose connective tissue separating the clusters of adipocytes.
2. High-power microscope observation, you can see the nucleus of the cell is flat round or semilunar, biased on the side of the cell.
(ii) Demonstrate the observation of Sudan Ⅲ (Sudan Ⅲ) stained flat slices of mouse mesentery.
The adipocytes are spherical and filled with fat droplets stained orange. Only a thin layer of cytoplasm remains and the nucleus is also compressed near the cell membrane.
Reticular organization
(i) Observation of sections (H-E stain) of lymph nodes from cats or rabbits
1. Low magnification observation The central part of the section with light coloring is the medulla, and the part with dark coloring surrounding the medulla is the cortex.
2. High magnification Observe the light reddish area of the medulla. The reticulocytes have multiple protrusions, which are connected to each other to form a network. The cytoplasm of the reticulocytes is abundant, the nucleus is large and the nucleolus is obvious.
(ii) Demonstrate the observation of silver-stained sections of cat lymph nodes showing reticular fibers.
Under high magnification microscope, the reticular fibers are black, uneven in thickness, and the branches are intertwined to form a net. Reticular cells are stellate, with protrusions, and neighboring cells contact each other with protrusions, which are also connected to form a mesh. Some cells with smaller nuclei and darker staining can also be seen on the section, which are lymphocytes in the lymph nodes, which do not belong to the components of the reticular tissue.
V. Cartilage
Observe with rabbit tracheal sections (H-E stained) to understand the structural features of hyaline cartilage.
(i) Naked eye observation
The blue-purple "C"-shaped structure in the wall of the trachea is hyaline cartilage, i.e. tracheal cartilage.
(B) Low magnification observation
In the hyaline cartilage, there is a blue-purple stained matrix and chondrocytes located in the traps. The chondrocytes in the central part of the cartilage were large, oval or rounded, and often existed in groups of two to four. Near the edge, the chondrocytes are smaller and denser, the cells are pike-shaped, and their long axis is aligned parallel to the surface of the cartilage. The cartilage is surrounded by a layer of dense connective tissue stained reddish color of the cartilage perichondrium.
(III) High magnification observation
Collagen fibers within the intercellular matrix could not be seen in the H-E stained sections. The stroma stained bluish-purple, with darker staining in the stroma around the traps. In some sections, due to the shedding of chondrocytes during the preparation process, a white cavity, i.e. cartilage trap, was revealed in the matrix. Some sections show white gaps around the chondrocytes due to contraction of some of the chondrocytes during the preparation process, which should also be part of the traps.
Bone tissue
Unstained transverse grindings of human bone are taken for observation. This is made by sawing thin slices from the bone-dense portion of a decaying femur and grinding them with a whetstone. The soft tissues (nerves, blood vessels, lymphatic vessels, connective tissue) and cells within the bone tissue have decayed, leaving only cavities and ducts, which appear black due to refraction of the light; the iridescent aperture of the microscope can be narrowed to make the light a little dimmer and clearer.
(I) Low magnification observation
Can be seen many bone plate was multi-layer concentric circles arranged structure, that is, the bone unit (Haversian system), each bone unit in the center of a black, larger circular tube cross-section that is the central tube (Haversian tube), in this tube around a number of concentric arrangements of the bone unit bone plate (Haversian bone plate).
In some cases, the connecting tubes between the central canal can be seen. If the bone fragments are taken intact, the inner and outer annulus plates and the penetrating canals that run transversely through the inner and outer annulus plates can also be seen. Between the bone units, some irregularly arranged bone plates, the interosseous plates, can also be seen.
(ii) High magnification observation
There are many small flat ovoid black cavities within or between the bone plates, i.e. bone fossa. There are many small black branches, i.e. bone tubules, emanating from the fossa to the surrounding area. It can also be seen that the tubules between neighboring fossae are connected with each other, and the tubules near the central canal are connected with the central canal.