Histology Methods Flashcards
What is the study of cytology?
Histology?
Organology?
Cytology – study of the cell
Histology – study of body tissues (4 primary tissues)
Organology – study of body organs (organs composed of 4 primary tissues)
What are the 4 primary body tissues studied in histology?
- Epithelium
- Connective tissue
a. Connective tissue proper
b. Blood and blood development
c. Cartilage and bone (both specialized connective tissues)
- Nerve tissue – composes both the peripheral and central nervous system
- Muscle tissue – consists of smooth, cardiac and skeletal muscle
What is the light (bright field) microscope used for?
– routine laboratory microscope used for studying tissue sections
What is a transmission electron microscope used for?
Transmission electron microscope (TEM) – used to study cytology or internal structures of cells; study of electron micrographs
What are scanning electron microscopes used for?
Scanning electron microscope (SEM) –used to study the surface features of cells and tissues; obtain a 3-dimensional picture of the tissue
What does a polarizing microscope allow one to do?
Polarizing microscope – permits one to determine whether biological materials have different refractive indices along different optical axes
What are phase microscopes used to study?
Phase microscope – used to study living tissue; works on principal of different refractive indices of cellular and sub-cellular components
What modification on the phase microscope allows for study of living tissue?
Interference micropscopy
What does fluorescence microscopy rely on?
uses UV light as the light source; used to examine the presence of fluorescent material in tissue sections
How does a confocal scanning microscope work?
Confocal scanning microscope – uses a laser energy beam; used to optically section a cell and with the appropriate computer equipment can reconstruct a 3-D image of the cell
In general, what are the 6 steps for preparing tissues?
- Fixation: preserve tissue morphology and chemical composition by rendering tissue insoluble by precipitating proteins and carbohydrates to stablize the structure
- Dehydration: uses alcohol to remove water from tissues so that tissue is miscible
- Clearing: replace alcohl with an agent miscible with paraffin (like toluene, xyelen or benzene)
- Infiltration and embedding: replace clearing agent with embedding material like paraffin, methacrylate, celloidin or gelatin
- Sectioning: produce thin sections throguh which light will pass using a microtome
- Staining: to impart color to a tissue
Besides fixation, how could one prepare a tissue?
What ttwo instances are these used for?
Use frozen sections!
Use for surgical boiopsies or for research studies looking at the localization of enzymes
- surgeon takes tissue sample for pathologist or tech
- Specimen is frozen and cut on freezing microtome
- Section is stained and examined
What are artifacts?
refers to any features evident in tissue sections that are a result of imperfect technique.
What are 6 causes of artifacts?
- Post-mortem degeneration – due to lysosomal digestion of the cells.
- Shrinkage – due frequently to reagents used in preparing paraffin sections, resulting in empty to clear spaces which during life were occupied by tissue components.
- Precipitates – occurs when formalin is not properly buffered.
- Wrinkles and folds – due to defect in paraffin section.
- Nick in the microtome knife – due to defect in knife, resulting in the tearing or scraping of the tissue when the section is cut.
- Mishandling of the tissue – frequently due to pinching of the tissue when removing tissue from the body.
Besides artifacts, what could lead to misinterpretation of a tissue section?
damage to the section
Why is staining necessary?
Living cells possess very little color, so sections of tissue are virtually colorless. This means we have to stain it if we want to see anything. Unfortunately the chemical basis of staining is poorly understood - we hae learned from trial and error through the years
What do acid and base stains do?
Basic stains stain cations (+)
Acid stains stain anions (-)
Note that this does NOT conform to the general understanding of an acid and a base.
What is a chromophore?
A chromophore is the atomic groups upon which color of a stain depends
What color is hematoxylin stain?
What does it stain?
Hematoxylin stain is blue to purple.
The stain has th chromophore associated with the basic radical, which is a cation, thus this is a BASIC stain. It will stain anything with a negative charge - a basophilic substance.
It will stain basophilic substances such as the nucleus, DNA, chromosome, heterochromatin, RNA nucleolus and cytoplasmic ribosomes (these all have POa 3-)
What color is the eosin stain?
WHat does it stain?
Eosin is red to pink
The dye has the chromophore assoicated with the acid radical - an anion, thus it’s an acid or anionic stain.
It will stain basic radicle - anything with a positive charge.
As such, it will bind acidophili substances like proteins (that have large numbers of basic groups assoicated with the side chains)
What will trichrome stain?
connective tissue, especially collagen
examples include Masson’s and Mallory’s stain
What will elastic stains stain?
Elastic fibers or elastic tissue in connective tissue
Examples include aldehyde fuschin, orcein, resorcin-fuchsin
What will silver impregnantion stains stain?
reticular fibers in connective tissue, also used in staining cells of the CNS
connective tissue fibers LOVE silver and will stain black
What are three examples of nucleic acid stains?
Fuelgen stain for DNA
?
What stain will color carbohydrates like glycogen?
periodic acid-Schiff reaction (PAS)
What 2 stains will find fats?
Oil red O (stains fats red)
Sudan black (stains fats black)
What used to be used for staining proteins?
What is used for staiing proteins now?
In the past, specific stains were used for specific AAs or groups of AAs
Today, we use immunocytochemistry to localize specific proteins in cells or tissues
What does immunocytochemistry allow for?
What are the steps involved in the procedure?
It premits the cellular or intracellular localization of specific proteins.
Steps:
- inject antigen into animal
- Animal produces antiboyd ot foriegn proteins
- Isolate the antibody
- Couple the antiboyd with a fluorescent compount like a peroxidase or ferritin
- Prepare the tissue
- Add the tagged antibody
- Wass off the excess and localize the tagged site in tissue section using fluorescent microscopy
What are the three different “tags” used in immunocytochemistry?
A fluorescent compound
Peroxidase
Ferritin/gold
What is the relative thickness of paraffin sections?
How about plastic sections?
paraffin: 6 to 7 micromilimeters
plastic: 1 to 3 micromilimeters
This is an unsteaind muscle tissue viewed through what kind of microscope?
a bright field microscope
This is an unstained muscle tissue viewed through what kind of microsope?
a polarizing microscope
This is tissue cultured cells viewed through what microscope?
phase microscope
These are actin bundles viewed under what microscope?
fluorescence
This is a thread cell viewed through what microscope?
light
Thread cell through which type of microscope?
transmission electron microscope
What type of microscope viewing a thread cell?
an electron microscope
The white portions of this section display what artifact?
post-mortem degeneration
The cells in this section display what artifact?
shrinkage
What artefact is displayed in this secion?
a fold or wrinkle
What is the difference between basic and acidic dyes?
Basic dyes have the chromophore in the cation, this the dye has a positive charge and will stain things with a negative charge (hematoxylin is an example).
An acidic dye has the chromosphore in the anion, thus the dye has a negative charge and will stain components with net positive charges (eosin)
Is Fast Green an acid or base dye?
How about methylene blue?
Fast Green acts as an acid cye because the chromophore lies in the anion of the salt
Methylene blud is a basic dye because the chromophore lies in the cation of the salt.
What stain was used to dye this small intestine section?
hematoxylin
What stain was used to dye this section of the small intestine?
Eosin
What was used to stain this section of the small intestine?
A combination of hematoxylin and eosin
What stain did this?
Trichrome stain
What stain did this?
Elastic fiber
What stain did this?
Reticular fiber stain
In general, how does the Fuchsin-sulfurous acid (schiff reagent) work as a stain?
Fuchsin-sulfurous acid (shiff reagent) is originally colorless, but when it interacts with 2 aldehydes, the product gives off a red color
How does the Feulgen reaction color DNA?
Tissue sections are exposed to mild HCl hydrolysis, which cleaves the purien bases from the deosxyribose sugars of the DNA, forming an aldehyde group.
Then, the section is incubated in Shiff’s reagent (which is originally colorless), which will bind to the exposed aldehyde groups, resulting in a red product
What stain did this? What specifically is stained?
This was done with through the Feulgen DNA stain.
Only the nuclei are stained
How does the periodic acid Schiff reaction work?
Incubating tissue sections in periodic acid will open up sugar to form aldehyde groups.
Schiffs's reagent can be applied to react with the exposed aldehyde groups to produce a red product
This is glycogen stained with what stain?
Periodic acid schiff (PAS) stain
What is this a PAS stain of?
kidney tubules: the basement and brush border
How does peroxidase activity allow for visualization?
- Incubate tissue with an antibody conjugated to peroxidate where the antibody is directed to come specific antigen within the tissue setion.
- Peroxidase will be localized to the vicinity of the antigen in question within the cell
- Incubate the section in substrates for peroxidase (like H2O2, and DAB,
- If the protein was present, black precipitates will form
Which stain was used for this section?
Peroxide stain
Specifically, the pars distalis of the pituitary gland was incubated in a solution containein antibody to grwoth hormone (conjugated to peroxidase).
