EEOB 3520 Exam 1
Basic stains - ✔️✔️like hematoxylin, which is a dye that has a net positive charge. It is
cationic. Hematoxylin will react with anion cells , basophilic substances, like carboxyl
end of a protein, nucleic acids ( dna, rna), glycosaminoglycan ( large sugar molecule),
nucleus in an acidic cytoplasm will stain blue because it binds to hematoxylin
What is clearing - ✔️✔️run sample through ↑ concentrations of alcohol then
infiltrate with solvent appropriate for embedding medium
- Clearing means you go from alcohol to something that is miscible in both alcohol and
wax, xylene or toluene( petroleum products that can dissolve wax)
What does fixation do? - ✔️✔️Fixation terminates metabolism, stops enzymes,
destroys pathogen, ( fresh brain is very soft, liquidy, fixation increases rigidity
What is formaldehyde? - ✔️✔️, formaldehyde is a gas, you don't preserve things in
formaldehyde, you have to put it in water, becomes formalin
What does formalin do - ✔️✔️formalin cross links proteins- forms bridges and prevents
them from being degraded
What happens after fixation? - ✔️✔️tissue needs to be dehydrated, clear it and prepare
to be embedded in medium to be sliced
Dehydration is necessary because tissue is 60% water, so need to remove water
How does embedding work? - ✔️✔️Material for embedding is wax, paraffin. Paraffin is
not easy to remove not water or alcohol soluble
Processing of tissue - ✔️✔️- machine goes up and down and agitates cassetes in bins
of liquids and rotates and drops down in a new bin
Then you end up with a piece of tissue in wax
How do you embed tissue? - ✔️✔️infiltrate tissue block with embedding medium
place in block, fill with liquid medium, and let solidify
There are molds that you put tissue in block in mold filled with melted wax
How do you section and mount tissue? - ✔️✔️use a microtome
5-15 μm thick
individual or serial sections
There is a blade and a block, evry time you turn handle the block advances, so little
slivers of block gets cut. They are so thin, they stick to each other
, After mounting you... - ✔️✔️dry, clear and rehydrate and stain
Sections are floated in warm water bath
Then mount it unto a slide
Then has to dry, and because tissue is thin, it is possible to reverse process used to
dehydrate, so need to put back into xylene and melt wax, then go in reverse order
through alcohol then finally in distilled water to rehydrate it, now you can stain it
Difference between histology vs microanatomy - ✔️✔️histology is the study of plant
and animal tissues
while microscopic anatomy is the analysis of cells, tissues, organs, and organ systems
Why use frozen samples - ✔️✔️rapid freezing is best. Freezing is faster than using
fixation. Some components of interest might be loss using the embedding process,
because alcohol dissolves fast.
use cryoprotectant for long-term storage - ice crystals are bad!
cut on cryostat (thin) or freezing microtome (thicker)
Acidic stains - ✔️✔️eosin, made up of acetic acid, dye part has negative charge,
anionic will bind to cationic groups in cells that are acidophilic. Will stain amino groups,
large proteins, collagen fibers. Looks pink
Other chemical stains can be used to specifically label - ✔️✔️carbohydrates
fibers/proteins
blood cells
muscle
Antigen - ✔️✔️non-cell protein
Causes immune response, body produces immune response against it. Proteins are
specific, because they are made by DNA
Immunostaining is based on this
Immunostaining and fluorescence microscopy - ✔️✔️don't need 2nd antibody. Just use
primary antibody with fluorescent tag on it. You can use different antibodies each with
different color tag
A cell must be able to - ✔️✔️obtain nutrients
eliminate waste
convert nutrients to energy
carry out biosynthesis
reproduce ( mitosis)
move/respond to environment ( Cells have to regulate what they are doing in response
to changes in environment)
