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What are the 5 steps of tissue preparation for a slide?
- 1. fixation
- 2. processing
- 3. embedding
- 4. cutting
- 5. staining
Why do we prepare tissue?
because if you don’t you know be able to see anything/much and it highlights the cells so its easier to determine what you are looking at
Why are staining techniques differ?
Sometimes you just need to see if something is there (such as detection of a microbe) and sometimes you need to actually visualize a structure
What stage of tissue preparation works to preserve the structure and integrity of cells and tissues and facilitates sectioning by making tissue more firm for this?
Common fixative (used for a standard biopsy)
10% neutral-buffered formaldehyde (formalin)
True/False: the fixative you use can have a big impact on your results such as it can give you false positive or negative
3 steps of tissue processing
- Specimen is dehydrated in an alcohol bath of increasing strength (70%-->100%)
- Specimen is bathed in xylene (clearing agent--washes the tissue after it has been dehydrated)
- Tissue is now ready for embedding in paraffin wax
What is used to cut the tissue and what is the standard thickness?
After the tissue is cut how is it placed on the slide?
It floats in a warm water bath and is picked up on a glass slide and is dried in the oven
What is the standard stain?
H&E stain (hematoxylin and eosin)
7 steps of staining and mounting
- parafin wax is dissolved from the slide (xylene)
- Tissue is rehydrated in alcohol baths of decreasing concentration (100-->70%)
- Stained (now automated except for frozen sections)
- Mounting medium (provides a better viewing for under the microscope
- Glass cover slip
This stain is a basic stain and so it stains acidic (basophillic) things
What color are acidic things stained when using hematoxylin, such as a nucleus?
purple or dark blue
This stain is an acidic stain and so it stains basic (acidophilic) things
What color are basic things stained when using eosin, such as esosinophils
pink or red
With Masson Trichome Stain what is stained:
What stain stains glycogen deep magenta?
Periodic Acid Schiff (PAS)
What are some things PAS is used for?
- Goblet cells
- Basement membranes (to check for damage) and brush border
- Some fungal organisms
- Used commonly in oral pathology and dermatopathology
What stains elastic tissue black?
Elastic Tissue stains (Verhoeff)
What is used to stain peripheral blood and bone marrow smears?
Wright Giesma Stain
Staining method that uses a specific antibody to bind to a specific antigen, such as a specific protein or an infectious agent
Positive "antigens" stain brown
Used in head and neck: lymph node, thyroid, salivary gland pathology
Aspiration cytology [fine needle aspiration biopsy (FNA)]
Used in oral cavity: candidiasis (most common way to rule out), white sponge nevus, oral hairy leukoplakia (infectious lesions that can affect size of tongue in immunocompromised patients), other
Classic example: pap smear
Exfoliative cytology (scraping of cells)
When you poke a tissue with a syringe and draw back some cells and use them
Not commonly used by pathologist because it is very tricky
Fine needle aspiration biopsy
Automated exfoliative cytology machine— should be able to determine if the cells are normal are not
**dont use this for malignant of premalignant cells because it is not as accurate
type of microscopy that allows person to look at cells at ultrastructrual level—looks more of like a standard slide
Transmission electron microscopy
type of microscopy that gives you a 3D image of little tiny thing just along the surface
Scanning electron microscopy (SEM)
When should you use SEM
when examining dental materials, look at surface of tooth, enamel
When should you use TEM
trying to see what is in the cell and structures
True/False: the way the specimen is cut, its orientation and where it is cut impacts how you see it in the microscope?
Examples in NOTES
what will you see if you cut a tube on the:
Transverse: cross section—will see rounded structure
Longitudinal: down the tube—will see two parallel lines of cells and maybe lumen
Example pictures in notes!!!
Type of intermediate filaments (6)
- Nuclear Lamins
What are microfilaments made of?
What are microtubules made of?
Tublin protein (alpha and beta)
What is the arrangement of the centrioles?
9 clusters of 3 microtubules
what are the microtubule arrangements in cilia?
9 clusters of 2 microtubules (doublets)
in a micrograph what differentiates the cilia from microvilli?
Basal body—structure at the bottom of the cilia
function of cilia
sweep debris, mucus, particulate matter, fluid, or cells across cell surface
True/False: microvilli do not move
function of microvilli
Increase surface area for absorption
transmembrane proteins involved in tight junctions that fuse together
What allows the adhering junction to stay together?
The transmembrane adherin proteins protrude from each cell and interdigitate
proteins are attached to actin filaments of the cytoskeleton
Consist of small channels called connexons and allow for the passage of fluid, small molecules, elections and ions between adjacent cells
What has Transmembrane adhesion proteins (cadherins) that are Bound to intermediate filaments (e.g. keratin)
True/False: pemphigus vulgarism affects the cadherin proteins and therefore affects the desmosomes and the hemidesmosomes
FALSE ONLY AFFECTS THE DESMOSOMES--NO CADHERINS IN HEMIS
True/False: all cells are actively dividing
Permanent (nerve and muscle cells)
Stable (liver cells)—stable until someone takes a chunk out, only sometimes dividing
Renewing (constantly dividing--skin, gastrointestinal, blood cells)
phase where there is Normal cell functions; duplication of organelles and increased cytosol
resting phase of interphase
Phase of interphase where DNA is replicated and histones are synthesized
phase of interphase where other centrioles are being replicated
Each replicated chromosome in the S phase=
2 sister chromatids attached by a centromere in the center
What is the difference between parietal and visceral pleura
The visceral pleura is the delicate serous membrane that covers the surface of each lung (the lung parenchyma) and dips into the fissures between the lobes. The parietal pleura is the outer membrane which is attached to the inner surface of the thoracic cavity. It also separates the pleural cavity from the mediastinum.
True/False: the visceral and parietal layers are continuous and one layer
Where do you find simple squamous?
- Alveolar spaces
Where do you find simple cuboidal?
- small glandular excretory ducts
- Renal tubules
Where do you find simple columnar?
- Digestive tract from stomach to anus
Where do you find pseudostratisfied columnar?
- Respiratory passages
- Reproductive (epididymus)
Where do you find stratified squamous?
Keratinized: skin, gingiva/alveolar ridge, palate
Non-keratinized: Oral (buccal mucosa, floor of mouth, ventral tongue, soft palate), esophagus, vaginal mucosa, anal mucosa (distal to anorectal junction)
Where do you find stratified cuboidal?
larger excretory ducts of salivary glands, pancreas, and sweat glands
Where do you find stratified columnar?
- conjunctiva of eye
- large excretory ducts
Where do you find transitional?
Bladder and urinary excretory ducts
**NOT kidney proper but once it leaves
What is this image illustrating and what stain is used?
A blood vessel with simple squamous cells
PAS? (periodic acid schiff)
Primary functions of simple cuboidal?
transport, absorption, secretion, excretion
Functions of simple columnar
What is this a picture of?
Simple columnar stomach cells
What are the main functions of pseudostratitfied
Function(s): Mucus secretion (Goblet cells), transport (ciliated columnar cells—like to move things around: debris, secretion)
What is in this picture?
Pseudo stratified cells in the nasal cavity
Simple columnar in the small intestine
simple columnar in the gallbladder
True/False: only stratified squamous cells can be keratinized or non keratinized
Other types of epithelium do NOT form keratin!
What is a metaplasia?
Transformation from one type of epithelial tissue to another
Form of keratin that has flattened nuclei in the keratinized cells and is more common in the oral cavity than skin
Form of keratin with no nuclei, keratohyaline granules present (stratum granulosum), basket-weave keratin (thin skin), and more commonly found in the skin than oral cavity
Parakeratinized cells in gingiva
Function of transitional cells
Protection, allows for stretch
Stratified cuboidal sweat gland
Stratified columnar in the conjunctiva
No excretory ducts. Secretions (hormones) are delivered to the bloodstream
Secretions delivered to a surface via excretory ducts
Example: mucous being produced in mouth or in respiratory tract
Only the secretory product is released from the cell (most common type)
What are examples?
Salivary, pancreas and sweat glands
The apical portion of the cell is released along with the secretory product
What are examples?
The entire cell is shed as the secretory product
What are examples
sebaceous (oil) glands
What are the characteristics of serous secretions (4)
Watery secretion often rich in enzymes
Dark-staining zymogen granules
Trapezoid-shaped cells with round, basally-located nuclei
What are the characteristics of mucous secretions (4)
Thick viscous secretion
Pale/white staining with frothy cytoplasm
Trapezoid-shaped cells with flat, basally-located nuclei
Lumen more patent
What is this a picture of and where?
- Left: Serous in the pancreas
- Right: Pancreas in the sublingual gland
What are the two types of exocrine cells?
- Unicellular (gobet cells)
- Multicellular (simple or compound and can have a duct and seculatory portion)
Three shapes of multicellular cells
- Tubular (test-tube like)
- Acinar/alveolar (flask like)
- Tubuloacinar (both)
What type of exocrine gland is the salivary gland?
One long tube that is coiled is very characteristic of ...
the sweat gland
Is coiled tubular compound or simple?
Simple because there is no compound coil
Pleomorphic adenoma in the parotid gland
Normal side is on the left because the architecture on the left is more normal and cell shape is more normal (white is fat)
The adenoma has some normal structure but also all those different cells in the background—most common salivary gland tumor (is benign but can undergo transformation)