Histology & Its Methods of Study: Preparation of Tissues for Study Flashcards
(26 cards)
Define Histology
the study of the tissues of the body & how these tissues are arranged to constitute organs
- involves tissue biology, focusing on how cells’ structures & arrangement optimize functions specific to each organ
What is the ECM and what does it do?
extracellular matrix
- supports the cells
- contains fluid transporting nutrients to the cells
- carries away wastes & secretory products
What happens to cells during development?
- they become functionally specialized with their associated ECM
- create distinct tissues with characteristic structural features
Define microtome
instrument used for sectioning paraffin-embedded tissues for light microscopy
- sections placed on glass slides,
allowed to adhere,
deparaffinized, and stained for
light microscopy
Define cryostat
- type of microtome in a cabinet at subfreezing temperature that is used to section the block with tissue
- frozen sections are placed on slides for rapid staining
- freezing of tissues used for very sensitive enzymes
- freezing, unlike fixation, does not inactivate most enzymes
Define Paraffin
- white/colorless soft solid hydrocarbon mixture derived from petroleum
- commonly used as embedding medium in histology to support and preserve tissue samples for microscopic examination
What is fixation?
- 1st step in tissue preparation
- small pieces of tissue placed in solutions
- chemicals that cross-link proteins and inactivate degradative enzymes for preservation of tissue and structure
Dehydration
- 2nd step in tissue preparation
- tissue transferred through a series of increasingly concentrated alcohol solutions
- ends with 100% ethanol to remove all water
Clearing
- 3rd step in tissue preparation
- alcohol is removed in organic solvents in which both alcohol + paraffin are miscible (mixable)
- replaces dehydrating agent (ethanol) with a clearing agent to make the tissue transparent
- preserves structure for accurate microscopic examination.
Infiltration
- 4th step in tissue preparation
- tissue placed in melted paraffin until tissue is properly infiltrated with the embedding medium (paraffin)
Embedding
- 5th step in tissue preparation
- paraffin-infiltrated tissue is placed in small mold with melted paraffin and left to harden at room temperature
Trimming
-6th (mostly unmentioned) step in tissue preparation
- paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome
Section Mounting
- trimmed tissue sections are mounted on glass slides for staining required to reveal specific cellular and tissue components with microscope.
- mounted on paraffin block holder
- each turn of drive wheel advances generally 1 - 10 um
Describe the H&E staining method
- Hematoxylin & Eosin
- most common staining method
- H stains DNA in nucleus, RNA-rich regions of cytoplasm, & matrix of cartilage dark blue/purple
- E stains collagen & other cytoplasmic structures pink
Define Anionic & Identify properties/associations
- describes cell components such as nucleic acids with a negative charge and an affinity for basic dyes
- basophilic
- DNA
- RNA
- glycosaminoglycans
- Hematoxylin
Define Cationic & Identify properties/associations
- describes cell components such as proteins with many ionized amino groups
- stain more readily with acidic dyes
- acidophilic
- mitochondria
- secretory granules
- collagen
- Eosin
Enzyme Histochemistry
lightly fixed or unfixed tissue sections to produce visible products in the specific enzyme locations.
- Fixation and paraffin embedding denatures most enzymes, so histochemistry usually uses frozen tissue sectioned with a cryostat.phosphatases, dehydrogenases, and peroxidases, with peroxidase often conjugated to antibodies used in immunohistochemistry.
Periodic Acid-Schiff (PAS) Reaction
- utilizes hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains such macromolecules purple or magenta
How can the DNA of cell nuclei be specifically stained?
using a modification of the PAS procedure called the Feulgen reaction
How can basophilic or PAS-positive material be further identified?
via enzyme digestion, pretreatment of a tissue section with an enzyme that specifically digests one substrate
- ex: pretreatment with ribonuclease will greatly reduce cytoplasmic basophilia with little overall effect on nucleus, making RNA important for this process
What is a less common method of staining and what does it entail?
- metal impregnation
- uses solutions of silver salts to visual certain ECM fibers & specific nervous tissue cellular elements
How are lipid-rich structures revealed?
- by avoiding processing steps that remove lipids (heat, organic solvents)
- staining with lipid-soluble dyes such as Sudan black and Oil Red O
What are lipid-soluble dyes like Sudan black useful for?
diagnosis of metabolic diseases involving intracellular accumulations of cholesterol, phospholipids, or glycolipids
How long does slide preparation, from tissue fixation to observation with a light microscope, take depending on the size of the tissue, embedding medium, and the method of staining?
12 hours to 2 1/2 days
