Kulkarni 1

We only had one lecture from Kulkarni so far due to a family emergency. LOOK AT HIS SLIDES!
Kulkarni Notes 1/3/07

Dr. Kulkarni teaches histology to medical students. This is his1st time to teach grad students. He runs a tumor bank, and has to look to see where normal tissue and where is cancerous tissue. We need some histology in order to learn pathology. We will see how to differentiate tissues. Will also explore how to process tissues and use them in research.
P1
Objectives and titles of 3 lectures.
P2
Human tissues cannot be sold in this country- get from a pathology department. Investigators need large number of cases. Pathology departments can supply the samples. They store FFPET for investigators to use for research.
Gross examination determines where tissue is normal or where it is not. After 10 years, the slides and paraffin blocks can be used for research.
P3-
Real case from the Med. Left- normal portion. Right-cancer. Colon. Take small portion and put in plastic cassettes. Fix in formalin so tissue becomes formalin-fixed. Then embed fixed tissue in paraffin.
P4
Can cut block on paraffin on microtome to get thin ribbon of formalin-fixed, paraffin-embedded tissue. Sections go on slides and are stained with different stains to visualize structures under the microscope. After ten years, the slides become highly valuable research material. Slides are used to generate a report for appropriate treatment of the patient.
P5
Formalin-fixed paraffin embedded tissue can now be used to extract analyzable RNA and peptides. Also used to immunostain for tumor markers.
Can take a type of cancer in different stages and stain for marker to se if it is helpful to diagnose cancer or not.
Last 5 years- tissue microarray has become possible. A small cylinder of tissue is taken from paraffin blocks. Take core with needle and put in recipient paraffin block. Recipient block has cores from different organs. May have 50-1000 organs. Section block and stain for marker. Saves a lot of money. Make these microarrays in pathology department.
Some chemicals used to fix specimens alter the markers used to identify cells. This is important to know if you want to research a tissue.
Tissue preparation:
Thin slices are made so light passes through.
P6
Fixation aim is to prevent autolysis and bacterial attack. Do not want to alter tissue or staining. Want to stabilize proteins along with other structures. If we look at cell, where nucleus is located is important.
Types of fixatives are listed.
RNAlater is an RNA stabilizing agent from Ambion. Commonly used to preserve RNA and used to prepare sections.
Formalin most commonly used.
P7
Formalin forms lysine-lysine crosslinks on outside of protein, not affecting structure and maintaining antigenicity.
Unbuffered formalin causes precipitation of formol-heme in tissue. How long to keep tissue in formalin- sections are usually 2 mm thick. Put in cassette for one hour because it penetrates from all directions. Volume of formalin should be 10x volume of tissue.
8
Tissue must be kept moist. If tissue dries, get artifacts to make differentiating normal and tumor tissue difficult. Formalin should be kept ready, but if not available, keep it moist.
Tissue stored in alcohol is good for investigating RNA.
9
After fixation, embed to take thin sections. Paraffin does not damage knife. Water in tissue must be replaced with other solution miscible with paraffin. Replace water with alcohol (dehydration), then with xylene (clearing), then with paraffin (impregnation-paraffin inside tissue) Then embed in paraffin. Embed- tissue with paraffin inside is put in block of tissue.
10
Paraffin block on microtome. Every time wheel rotates, block moves forward, down and up. Get ribbon of sections and put on slide and stain with H and E.
11
How cells look under microscope
We will learn how normal cells look.
Can see cell boundaries and nuclei clearly. This is H and E stain. Can see nucleus, nucleolus, and cytoplasm.
We have to choose different cells to see different organelles using different techniques.
12
There are normal cells with multiple nuclei. Sometimes in liver you get two (center)nuclei in a cell. Left-skeletal muscle. Nuclei at periphery are flattened. Liver- one or two nuclei in center. Right- neuron with prominent nucleus off slightly to one side and prominent nucleolus.
N/C ratio compares ratio of nucleus to cytoplasm. Ratio disturbed in cancer. Nucleus gets bigger.
Nucleus
Nucleus on left contains clumps of chromatin because it is in interphase, Cell undergoes division- you will see chromosomes. Chromatin is like tightly coiled spring. Whole thing tightly coiled in chromosomes. Some parts coiled- chromatin. Chromatin is found in resting or interphase. The uncoiled portion produces protein- euchromatin. Not visible. Tightly coiled is visible- not making protein.
Cell not very active- nucleus stains darkly. Vesicular nucleus does not stain darkly.
13
Special features help us identify cancer.
Groove or fold on nucleus- papillary carcinoma.
Sometimes divided in lobules.
Right photo: Barr body is what arrow is pointing to. Seen in female nucleus. 46 chromosomes with 2 X chromosomes. Males have one X and one Y. In females one X chromosome is inactivated and becomes heterochromatin. More than 2 X chromosomes- get another Barr body.
Left- nucleus has chromatin at periphery with one part at center- found in plasma cell. Clock face heterochromatin.
Autophagy- smooth ER surrounds a worn-out organelle and tries to digest it.
14
Debris-filled vacuole seen as lipofuschin or age pigment- seen in cardiac muscle.
Seen also in liver.
Plasma cell in picture is oblong with nucleus on one side with clock-face chromatin. Unstained portion next to nucleus is the Golgi apparatus and the rest is pink. This clear, unstained area is the perinuclear hof.
15
Left skeletal muscle- see striations because of actin and myosin.
Desmosomes anchor cells to one another or to basal membrane.
Right- cilia from basal bodies. Center- microvilli. Cilia are hair-like, villi are finger-like. Intermediate filaments can be immunostained. Types are tissue-specific.
16
Communicating junction- 2 cardiac muscle cells have a gap junction or nexus between them. Intercalated disks contain gap junctions. Left- terminal bars are cell junction anchoring cell together.
Inclusions can be seen.
Upper right- cell with lipid droplet in middle. Lipid dissolves in alcohol and xylene, so you get empty space . Cell gets signet ring appearance due to empty space and flattened nucleus over to side.
Zymogen seen in salivary glands on lower left. Nucleus over to one side. Mucinogen granules dissolve in water during processing and leave foamy or soap-bubble appearance (middle-right of lower left). Lower right- goblet cell.
17
Tissues
Epithelial – covering, lining, glands. Cells toward base rest on amorphous basal membrane. Trachea has thickest basement membrane.
18
Transitional epithelium= some cells not on basement membrane. Toward lumen have umbrella cells. Considered transitional epithelium or urothelium. Seen when bladder is empty. Filled with urine- all layers get flattened. Transitional epithelium changes shape according to conditions of organ.
Also classified by shape of surface cells. Cells toward surface in esophagus are facing lumen. On skin, face body surface.
Squamous means flat cells. No real height.
Cuboidal –shaped like cubes.
Columnar- tall.
19
Simple squamous- most of body cavity lining. Called mesothelium from development.

20
Pseudostratified epithelium falsely appears to be multilayered. Some cells are smaller or larger. See two rows of nuclei, which look like layers. Actually simple epithelium.
Stratified squamous- multilayered and cells toward surface or lumen are squamous. Cells toward surface are flat.
Keratinized- cells form amorphous layer of keratin (middle) protective layer in center.
Right- esophagus- no keratin on top.
21
K/as in notes means “known as”
Top picture is stratified columnar.
Bottom- left- cuboidal to pear shape to umbrella cells. They form continuous lining on urinary bladder. Left empty. Right is full.
22
Cilia propel mucus. They start from basal body. White arrow on left- goblet cell.
Microvilli fingerlike projections increase surface area multifold.
Stereocilia are false cilia – really long microvilli.
Glands
Epithelium invaginates into connective tissue during development. Forms bulb at end and bulb forms gland. Connecting stalk forms duct. Sometimes ducts disappear to form endocrine glands. Gland gets surrounded by blood vessels and secretes into blood. Exocrine have a duct, like salivary gland