Histological Staining and Cell Structure

 


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Cell structures made visible by staining with hematoxylin and eosin

 

The combination of hematoxylin and eosin is the most commonly used method to stain sections of tissues.

Hematoxylin, a basic dye, binds to acidic components of a tissue, which are thus said to be "basophilic." The color of the stained structures depends on the mordant used to make the hematoxylin dye bind to the molecules of the tissue. Potassium alum, the most common mordant, gives the dye a blue to purple color.

Eosin, an acidic dye, binds to basic components of a tissue, which are thus said to be "acidophilic." The structures stained by eosin are typically colored pink to red.

 74

Examine a section of salivary gland stained with hematoxylin and eosin. Identify the following structures in the section of salivary gland :

  1. Nucleus
  2. Nuclear envelope (basophilic due to adsorbed DNA)
  3. Nucleolus (how many per nucleus?) (basophilic due to DNA and RNA)
  4. Chromatin (basophilic due to DNA)
  5. Cytoplasmic RNA (basophilic) - present in secretory cells but not in duct cells
  6. Cytoplasmic protein (acidophilic) - see duct cells
  7. Cell outline
  8. Mucus - does not stain with hematoxylin or eosin

67

Soft palate. Identify the same strutures.

 suppl.

Pituitary gland. Identify the same strucures.
suppl. Duodenum. Shows nuclear and cytoplasmic staining.

94

Anal canal. Identify the same structures.
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 Cell structures visible because of their shape or pigmentation

  Some cellular structures can be identified in the light microscope because they have unique shapes or because they are naturally pigmented. Identify the following structures in the slides indicated:

 123

130

suppl.

Chromosomes, here stained with hematoxylin, are large bodies found in mitotic and meiotic cells.

 130

Mitotic spindle, composed of microtubules, which are not visible as individual structures in the light microscope.

 130

Centrosome - site of the centrioles (too small to resolve) and the region where the spindle microtubules come together at opposite poles in the cell.

102

Zymogen granules - spherical vesicles containing secretory proteins in pancreatic acinar cells. They stain pink with eosin.

 108

Cilia - elongated motile extensions from cells containing a core of microtubules, seen here on the interior surface of the trachea.

 123

suppl.

Flagella - slide 123. A single flagellum, containing a core of microtubules, forms the tail end of sperm.

 62

suppl.

Melanin pigment granules, which can be seen in sections because they are naturally yellow, brown or black in color, impart a dark color to the tissue that contains them.

 124

suppl.

Stereocilia are elongated microvilli containing a core of actin microfilaments. They are seen here on the interior surface of the epididymis.

 Structures made visible by special stains

  Other cellular structures can only be identified in the light microscope if special stains are used. Examine the following slides, noting the different appearance of the tissue caused by different staining procedures and identify the structures listed.
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Impregnation of tissue with silver

  Silver is added to tissues in an ammoniacal solution as Ag (NH3)+ and subsequently reduced to form metallic silver. The precipitated silver appears black in the light microscope and the specificity of staining can be determined by controlling the composition of the staining solution and the conditions of the reduction reaction.

 6

Bielschowski's silver impregnation method was used to stain the reticular fibers in this human lymph node. Nuclei and other structures are stained black-brown. Reticular fibers are produced by cells, but they are part of the extracellular matrix.

 1

Ammoniacal silver nitrate was used in this preparation of mesothelium to deposit silver along the boundaries of the covering epithelilal cells. It is thought that the silver binds to cell surface molecules.

 102a

In this preparation of pancreas, silver was precipitated in the vesicles of the Golgi apparatus prior to sectioning, staining the Golgi black. Compare this section to the companion section on the same slide stained either with iron hematoxylin and eosin, which stains the zymogen granules pink 102, or iron hematoxylin, which stains the zymogen granules black.

38

39

Silver nitrate was used to stain the nerve cells in these sections black. The gold background color is an artifact of the method and not due to a counterstain.
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 Osmium

33

33a

Osmium is another metal used to stain cells. Osmium in the form OsO4 (osmium tetroxide) is a lipophilic stain and, therefore, stains lipid droplets and membranous structures black. In combination with potassium dichromate, osmium specifically stains entire neurons.Osmium stained sciatic nerve of the frog. Osmium stains the myelin sheath black.

 Iron Hematoxylin

 

 

Hematoxylin, when combined with iron as a mordant, can be used alone to bring out structures not clearly delineated otherwise, including mitochondria, muscle fiber striations and the myelin sheaths of nerves. It is also an excellent stain for cell nuclei (stains chromatin black) as in Weigert's iron hematoxylin stain.

22

suppl.

Skeletal muscle. The striations are caused by the presence in the cells of aligned thick and thin filaments composed of myosin and actin, respectively. Similar filaments are found in almost all types of cells, but they are too small to be resolved in the light microscope. In muscle, the individual filaments are not resolved, but the striations are the direct result of large numbers of filaments being aligned in register. (See appropriate figures in tests).

76

IH-stained mitochondria in salivary gland. Use oil immersion to resolve the black spherical and elongated structures.

101

IH-stained mitochondria in the pancreas. 

 Aniline Blue

  This stain has an affinity for collagen, cartilage matrix, and mucus. Therefore it is frequently used to differentiate these components in sections that have already been stained with other dyes. It is most often used as a counter stain with iron hematoxylin (IHAB).

 Iron hematoxylin and Aniline Blue (IHAB)

 

The combination of analine blue (acid stain) with iron hemetoxylin (basic stain), often abbreviated as IHAB, is very effective for demonstrating the collagenous connective tissues. Nuclei and other basophilic structures are stained black to brown, whereas collagen fibers are stained bright blue.
111 Kidney stained with IH and aniline blue. Nuclei and other basophilic components are stained black with IH. Collagen fibers of the connecive tissue and the basement membrane are counter-stained blue with aniline blue.

 73

Submaxillary gland. This section is stained with a combination of iron hematoxylin (stains chromatin and cytoplasmic ribosomes black to brown) and aniline blue, which differentiates the connective tissue septa of this gland.
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 Azocarmine and aniline blue (AZAN)

 

In this combination of the basophilic dye azocarmine with aniline blue, nuclei and basic structures are stained red and collagen, mucus, and cartilage matrix are stained blue.

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Fibrocartilage. Note the red-stained nuclei and the blue-stained conective tissue.

 112

Kidney. Note the red-stained nuclei and cytoplasm (basophilic) of the kindey cells and the blue-stained basement membrane.

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Spinal cord. Note the red (purple) stained cells and blue stained fibers of the connective tissue (arrow).

 Hematoxylin and Potassium Iodide (Verhoeff's elastin stain)

  With KI as a mordant, hematoxylin stains the elastin fibers of connective tissue purple to black.

 2

Areolar (loose) connective tissue stained with Verhoeff's stain and counter-stained with ponceau S, a reddish acid dye. Note:

  1. elastin - black (fine lines)
  2. nuclei - blue-purple
  3. collagen - red-pink

 13

Elastic cartilage. Note the elastic fibers, which stained purple-black.

Cresyl Violet (Nissl stain)

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Nissl stain stains rRNA purple. The so-called Nissl substance in the cytoplasm of nerve cells has been identified as ribosomes and the rough endoplasmic reticulum. This stain gives the Nissl substance a purple color.Find the giant motor neurons of the spinal cord. The nucleus appears light in color, almost unstained, containing a large prominent nucleolus, stained purple because of its high content of rRNA. The dark purple staining of the cytoplasm is due to ribosomes, which also contain rRNA.
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 Azures

  The azures are a family of related basic dye compounds that are often used in combination with acidic eosin to produce contrasting red and blue stained tissues. One such dye mixture is the commonly used Wright's stain for blood smears. Another is Giemsa stain. The variety of effects that can be produced by combining azures with other dyes are demonstrated in the following slides.
Azure alone

 3

Mesentery. Azure blue stains cell nuclei blue and mast cell granules metachromatically purple.

 Azures and Eosin (Wright's stain)

 54a

54b

54c

54d

Human blood, stained with Wright's stain, shows:

1) erythrocytes - pink-red

2) leukocytes
a) cytoplasm - unstained-pale blue
b) nuclei - dark blue-purple
c) eosinophilic granules - bright red, large
d) neutrophilic granules - pale purple pink, small
e) basophilic granules - deep blue-purple
 

3) platelets - lavender

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Mallory's Triple Stain

16

This is one of several commonly used techniques in which three or more dyes are combined. These multiple-dye stains have the advantage of showing a larger number of tissue structures. Mallory's Stain combines aniline blue, orange G (stains proteins) and acid fuchsin (stains DNA and RNA). This slide shows the formation of membrane bone. In this slide collagen-containing connective tissue is blue, erythrocytes are orange, and chromatin, nucleoli, basophilic cytoplasm, and muscle cell cytoplasm are red.

 Luxol Blue - Periodic Acid- Schiff - (Hematoxylin)

105a

105b

In this combination stain, Luxol blue stains lipids and lipo-proteins blue-green and PAS stains carbohydrates rose to red. In the absence of hematoxylin, nuclei are blue, but adding hematoxylin makes the nuclei dark (basophilic).Mouse gut. One section is without hematoxylin 105a; one is with hematoxylin 105b.
Hematoxylin, Phloxine and Saffron This combination of three stains is used mostly to demonstrate collagen in connective tissues. Hematoxylin, a basic dye, stains acidic structures such as DNA, purple. Phloxine, an acidic dye, stains basic structures including most proteins, pink. Saffron stains collagen yellow.
109 Mammalian lung, bronchus. Note how the relative intensity of yellow staining, due to the saffron, demonstrates the relative concentration of collagen in different parts of this section through the wall of a bronchus.




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