Histological Methods & Cell Biology Flashcards
what are the two primary methods of tissue preparation
Fixation and fresh frozen
How is tissue fixation done?
Tissue is treated by chemical or mixture of chemicals to permanently preserve the tissue structure. Most common is formalin. Process takes several hours to days
What are the reasons fixation might be used?
cell metabolism ceased
enzymatic degradation prevented
pathogenic organisms killed
tissue is ‘hardened’, i.e. integrity strengthened
What happens after fixation?
After fixation tissue is embedded in a support matrix. This enables cutting thin (5-15um) sections onto glass slides. Tissue is cleared of support matrix. Tissue is stained.
How is fresh frozen tissue prepared?
Tissue is frozen (-50°C) Tissue is cut (sectioned) onto glass slides inside a cryostat at freezing temperatures. Tissue is stained Process takes minutes
When are fresh frozen tissue samples frequently used?
Frequently done for surgical samples. Part of the sample is fixed and analyzed further to confirm diagnosis from fresh frozen sample.
How do stains/dyes work?
React to the chemical properties of the molecules in the tissue such as net electrical charge (electrostatic interactions)
How do acidic dyes work? (Eosin)
They carry a net negative charge - reacts with cationic groups (carry a net positive charge)
Cationic groups are considered…
Acidiophilia
Acidic dyes work well with what cellular structures?
cytoplasmic filaments (muscle cells)
intracellular membrane components
extracellular fibers (ionized amino groups)
How do basic dyes work?
they carry a net positive charge - reacts with anionic groups (carry a net negative charge)
Anionic groups are considered…
basophilia
Basic dyes work well with what cellular structures?
Phosphate groups of nucleic acids
- heterochromatin & nucleoi (nucleotides)
- cytoplasmic components (rRNA)
ionized sulfate groups of glycosaminoglycans
- extracellular materials
Carboxyl groups of proteins
What does H and E stain stand for?
hematoxylin and eosin
When are H and E stains usually used?
most commonly used stain to reveal structural features of the tissue
Is hematoxylin a true basic dye?
No. It relies on mordant which acts as a link between the dye and the tissue
What is a PAS reaction
Periodic acid-Schiff reaction
What does a PAS reaction do?
It identifies carbohydrates and carbohydrate rich molecules (glycoproteins) Glycogen Basement membrane Reticular fibers
What is immunohistochemistry?
Specific interaction between antibody and antigen
Direct immunofluorescence
A fluorescent antibody directly binds to the antigen and then begins to glow
Indirect immunofluorescence
A primary antibody first binds to the antigen, then 2+ fluorescent secondary antibodies bing to the primary antibody and begin to glow.
What is hybridization?
Specific interaction between nucleotide (DNA, mRNA) and complementary nucleotide strand
What is resolution?
The minimum distance 2 objects are separated and seen as 2 object
What is the Resolution equation?
r = distance two objects are from each other
λ = wavelength of light
NA = numerical aperture of the objective lens that collects light
n = index of refraction of the medium between object and lens
How does bright field, phase-contrast, and differenetial light microscopy work
Bright field: light passes through, requires stain to see specimen
Phase-Contrast: differences in refractive index reveal dense (dark) parts of specimen
Differential: interference (surface properties)
Transmission EM vs Scanning EM
Transmission EM: Heavy metal stains give light or dark, Dark areas are electron dense (scatter electrons), Tissue is sectioned
Scanning EM: Thin metal coating. Not sectioned, surfaces are scanned
what is fluorescence microscopy
an optical microscope that uses fluorescence to study the properties of organic or inorganic substances
What is Confocal microscopy
confocal microscope uses fluorescence optics. Instead of illuminating the whole sample at once, laser light is focused onto a defined spot at a specific depth within the sample. This leads to the emission of fluorescent light at exactly this point.
What are the components of the nucleus?
chromatin (heterochromatin & euchromatin), nucleoli, nucleoplasm
Nuclei that have a high amount of euchromatin indicate…
indicate high level of protein synthesis needed to maintain metabolically active cells
= high level of constant gene transcription.
3 distinct regions of nucleolus
and the components of the nucleolus
Fibrillar centers (nucleolar organizer; NO) – DNA loops
Fibrillar material (pars fibrosa; PF) – ribosomal genes & rRNA
Granular material (pars granulosa; PG) – initial ribosome assembly
Consists of portions of 5 pairs of chromosomes that code for ribosomal RNA (rRNA)
Components of the Nuclear envelope: Bilayer, Outer membrane, Inner membrane, Nuclear lamina
Bilayer membrane encircling the nucleus.
Outer membrane layer of nuclear envelope is continuous with rER.
Inner membrane layer of nuclear envelope is lined by nuclear (fibrous) lamina consisting of lamins A, B & C (intermediate filaments) that form a ‘lattice’.
Nuclear lamina provides structural support and attachment sites for chromatin.
Composition of the Nuclear pore Complex (NPC)
composed of 50 different proteins called nucleoporins that form 70-80nm diameter openings in the nuclear envelope
What are the requirements of large molecule transport through nuclear pores?
nuclear localization sequence (NLS) to bind cytoplasmic importins to shuttle it to the NPC.
nuclear export sequence (NES) to bind nuclear exportins to shuttle it to the NPC.
What are the requirements of small molecule transport through nuclear pores?
cross by simple diffusion (energy independent) via water-filled channels
What do mRNAs move from nucleus become associated with?
membrane-associated polysomes in rER
cytoplasmic ‘free’ polysomes
intermediate compartment of sER
Golgi
What are the cellular components of Endoplasmic reticulim
ER is a continuous array of membrane bound tubules, vesicles and cisternae.
Rough ER (rER) and Smooth ER (sER)
rER is studded with ribosomes (polysomes)
rER is continuous with the outer membrane of the nuclear envelope.
What Postranslational modifications that occur in the rER
Glycosylation
Disulfide bond formation
Hydrogen-bond formation
Folding
What are the functions of the sER?
* Detoxify and modify hydrophobic compounds - convert to water-soluble forms
* Lipid and steroid metabolism
* Glycogen metabolism
* Membrane formation and recycling
What is the Smooth ER called in skeletal and cardiac muscle where it is involved in Ca2+ regulation?
Sacroplasmic reticulum
What are the functions of the golgi apparatus?
Postranslational modification of N-linked oligosaccharides
Sorting and packaging of proteins
Sorting signals on the proteins ensure proteins are sent to:
Apical plasma membrane (extracellular and membrane
proteins)
Basolateral plasma membrane (membrane proteins)
Endosomes or lysosomes (organelle proteins)
Apical cytoplasm (secretory proteins)
How are the vesicles leaving the TGN delivered to the correct destination?
by sorting signals and/or physical properties of the proteins being transported
What are the 3 networks of the golgi apparatus
The cis-Golgi network is closest to the rER, then medial- Golgi network, and then trans-Golgi network (TGN).
Proteins exit Golgi from the TGN in vesicles
What is pinocytosis?
constitutive, nonspecific, small particles
what is phagocytosis?
ingestion of large particles, receptor mediated, primarily by mononuclear phagocytotic system
What is receptor mediated endocytosis?
specific molecules ingested (cargo receptors bind to cargo proteins), clathrin-dependent (coated pits)
Early vs Late Endosomes
Early endosomes sort and recycle internalized proteins
Late endosomes mature into lysosomes
What are lysosomes? what are they composed of?
Are digestive organelles
rich in hydrolytic enzymes (proteases, nucleases, glycosidases, lipases, phospholipases)
What are lysosomal membranes resistant to?
lysosomal membrane resistant to hydrolytic enzymes
pH is low (4.7) due to proton (H+) pumps transporting H+ into lumen
Where are lysomal proteins made?
made in the rough ER.
are targeted to the lysosome by specific signals such as M-6-P (mannose-6-phosphate
what are the functions of mitochondria
ATP production
regulate cytoplasmic ion concentration (Ca2+)
What is apoptosis and when does it occur?
programmed cell death
occurs under normal conditions, external and internal stimuli regulate:
Loss of mitochondrial function,
Bcl2 family of proteins – mitochondria release cytochrome c - activate caspases
TNF receptor – ‘death receptor’ – external stimulus
What is necrosis?
Necrosis is a pathological process that results from cellular injury
What is the function of the cytoskeleton? What 3 polymers make up the cytoskeleton?
Cytoskeleton is the basis for cell morphology and plasticity
Three primary components:
- Actin filaments
- Microtubules
- Intermediate filaments
What is the cellular role of actin filaments?
Anchors at cell junctions
Structural core of microvilli
Cell locomotion
Extension of cellular processes
Describe the structure of actin filaments
helical arrangement of actin monomers (i.e. polymerized actin; F-actin). Actin monomers (free actin) are globular (G-actin)
Polar structures
Dynamic (barb and pointed ends) 4-6nm diameter, 400-800nm long
Where is actin most abundant?
in subplasmalemmal cortex and lamellipodia of migrating cells
Describe the structure of microtubules
helical cylinders composed of pairs of alpha and beta tubulin
nucleated by y-tubulin in MTOC (microtubule organizing center; centriole)
Polar structures
Dynamic (+/– ends, fast and slow growing)
Hollow tubes, 25nm diameter, length varies
What is the role of microtubules?
Crucial roles in Intracellular transport, cell morphology, cell division, flagella movement
Describe the structure of intermediate filaments
Coiled ‘ropelike’ fibers, 5 classes of proteins compose (keratins, vimentin & vimentin-like, neurofilaments, lamins, beaded filaments)
- Lack enzymatic activity and are not polar
- Display a degree of stability
- Diverse and tissue specific
- 8-12nm diameter, many ums long
What is the role of intermediate filaments?
Crucial roles in stabilizing and maintaining cell morphology
Describe the structure of centrioles
Cylindrical array of nine triplets of microtubules surrounding a lumen - pinwheel
Oriented at right angles
What are the roles of the centrioles?
Basal body for assembly of cilia & flagella
Align mitotic spindle during
cell division
Nucleate microtubules
What are the 3 families of cytoskeletal motor proteins?
Myosin family
Dynein family
Kinesin family
Describe the structure of molecular motor proteins
- ‘motor domain’→ convert ATP via hydrolysis into mechanical force
- tail → self-associate and/or bind to particular cargo(cytoplasmic vesicles, membrane-bound organelles, chromosomes, etc.)
Myosin family
What cytoskeleton polymer does it use?
only molecular motors that use actin filaments
myosin II (in muscle cells actin+myosin = 60% total protein)
