Introduction and Epithelium Histology Lab 1 Introduction and Epithelium Jun Zhou（周俊）, Ph.D & M.D School of Medicine,Zhejiang University 20120924

Note: Please come lab on time. Find your seat according your lab number.

Your final score is composed of four parts 1) Attendance and picture drawing:10% 2) Quiz: 15% (each quiz 5%) 3) Final lab test:25% 4) Final written examination: 50%

Lab facilities: 1 Light electron microscope with computer 1 case of slides 1 atlas 1.Students share these facilities 2.Do not take them out the laboratory with you. 3.Put them where they belong to when lab is over. 4.Do not left any rubbish after you.

Contents of Lab 1: Introduction Epithelium 1)Simple squamous epi (small intestine,stomach) 2)Simple cuboidal epi.(thyroid) 3)Simple columnar epi.(small intestine)(drawing) 4)Pseudostratified ciliated columnar epi.(trachea) 5)Stratified squamous epi.(esophagus) 6) Transitional epi.(bladder) 3.Lab report (content of lab , drawing)

Tissue Processing 1.Fixation: 10% Formalin 2.Dehydration:graded concentrated ethyl alcohol 3.Clearing:xylene,infiltrate with paraffin solvent. 1. Fixation: (Dorland p. 682 ‘fixative’) - To preserve the tissues in a state which can be correlated with the conditions in the living patient - Most common fixative is 4% buffered formaldehyde (Formaldehyde is available commercially as a solution in water marketed as “formalin”, so most pathologists will tell you they use “10% formalin.” This is effectively a 4% formaldehyde solution.) - Other fixatives are available depending on what structure is to be examined 2. Dehydration & clearing - Water in tissues is removed by placing tissue in a series of alcohol baths (Usually isopropyl or ethyl alcohol in baths going from 70% to 100% alcohol) - Alcohol must be removed (“cleared”) from the tissues, since alcohol is immiscible (won’t mix with) most embedding agents - Xylene was once the most common clearing agent; most labs now use substitutes that are less dangerous - Removes neutral fats from tissue (thus fat cells look like great big empty spaces with nuclei) 3. Infiltration & Embedding - Infiltrating the tissue with melted paraffin takes several hours, usually overnight, depending on size of tissue involved - Embedding: Infiltrated tissue fragments are placed in a mold (‘block’) with more paraffin, which allows proper orientation of the specimen Loss of fat due to ‘clearing’

Tissue Processing 5.Sectioning 4.Embedding 3. Infiltration & Embedding - Infiltrating the tissue with melted paraffin takes several hours, usually overnight, depending on size of tissue involved - Embedding: Infiltrated tissue fragments are placed in a mold (‘block’) with more paraffin, which allows proper orientation of the specimen 4. Sectioning - A microtome is used to cut reproducibly thin section of tissue - The very thin slices of tissue (2-5 microns thick) allows the transmission of light through the tissue

Tissue Processing 6.Staining

Common Stains: Hematoxylin & Eosin Ross, 5th ed. Fig 1.1 Hematoxylin and eosin (H&E) staining. This series of specimens from the pancreas are serial (adjacent) sections to demonstrate the effect of hematoxylin and eosin used alone and hematoxylin and eosin used in combination. A. This photomicrograph reveals the staining with hematoxylin only. Although there is general overall staining of the specimen, those components and structures that have a high affinity for the dye are most heavily stained, e.g., nuclear DNA and areas of the cell containing cytoplasmic RNA. B. In this photomicrograph the counterstain, eosin, likewise has an overall staining effect when used alone. Note, however, that the nuclei are less conspicuous than in the specimen stained with hematoxylin only. After the specimen is stained with hematoxylin and is then prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost, and the eosin then stains those components to which it has a high affinity. C. This photomicrograph reveals the combined staining effect of H&E. Ross, 5th ed, fig 1.1

Common Stains Hematoxylin and eosin ("H&E") Wright’s stain From Bancroft, Histologic Techniques, p. 96: Acid & basic dyes are not acids & bases, but salts whose coloured species are anionic and cationic respectively. Neutral dyes are not non-ionic but are salts in which both the anion and cation are dyes. P. 99: Haematoxylin is extracted from the heart wood (‘logwood’) tree Haematoxylin campechianum from Mexico/West Indies. It is not a stain but its oxidation product haematein is the dye. Haematein is anionic & has poor affinity for tissue & requires a ‘mordant’, the combination of which gives a net positive charge & allows it to bind to anionic tissue sites. 1. Hematoxylin and eosin ("H&E") - The most common routine stain - This results in a pattern of blue & pink (or orange) a. Blue (hematoxylin): Materials which take this stain are called “basophilic” - Cellular components which have a high proportion of nucleic acids are generally basophilic (nucleus, ribosomes) - Calcium deposits tend to be basophilic b. Pink/Orange (eosin): Materials which take this stain are called “acidophilic” or “eosinophilic” - Mitochondria, plasma membranes, smooth endoplasmic reticulum are usually eosinophilic - Extracellular materials such as collagen are usually eosinophilic, although less so than intracellular organelles Wright’s stain

Relative Sizes in Histology Standard Units of Measure Remember the standard units of measure? S.I. (standard international) units micrometer - 10-6 meter, also called micron, sometimes . nanometer - 10-9 meter, sometimes called millimicron Ångstrom (not a S.I. unit) - 10-10 meter

Relative Sizes in Histology Standard Units of Measure nanometer: 10-9 meter micrometer: 10-6 meter Remember the standard units of measure? S.I. (standard international) units micrometer - 10-6 meter, also called micron, sometimes . nanometer - 10-9 meter, sometimes called millimicron Ångstrom (not a S.I. unit) - 10-10 meter [Alberts p. 549 has a nice set of cartoons illustrating relative sizes; also p. 551 diagram of relative scales (fig 9-1 & 9-2)] RBCs and many normal nuclei are about 7 micrometers in diameter Cilia on respiratory epithelium are about 2-3 microns in length (Junqu) cross section of a microtubule is about 25 nm (Cross p. 34)

Changes in apparent shape, due to orientation of sections Ross, 5th ed, fig 1.11

Changes in apparent shape, due to orientation of sections Netter/Ciba Junqueira fig 1-30

Epithelium No blood vessles, nourishment is by diffusion Epithelium is innervated polarization Trachea

Classification of Epithelia Number of cells 1. Simple 2. Pseudostratified 3. Stratified Shape of surface cells 1. Squamous 2. Cuboidal 3. Columnar 4. Transitional

Stomach (NO 25): mesothelium HE ×400 Simple squamous epi.

Small intestine (NO 1): endothelium HE × 400

Thyroid (NO 35): HE × 100

Thyroid: HE × 400 Simple cuboidal epi.

Small intestine (NO 1): villus HE × 400 (Drawing ) Simple columnar epi., Goblet cells, striated border (microvilli)

Trachea (NO3): HE × 100

Trachea: HE × 400 Pseudostratified ciliated columnar epi.

Esophagus (NO2): HE × 40 Stratified squamous epi.

Esophagus: HE × 100

Bladder (NO33): HE × 40

Bladder: HE × 100

Bladder: HE × 400 Transitional epi.

The end!