module 3 Flashcards
what is histology
study of microscopic anatomy
what is tissue fixed with for light microscopy
4% formaldehyde in phosphate buffer that cross-links proteins so tissue does not degrade
what did we stain rat brain tissue with in lab
cresyl violet stain
how was the rat brain in lab fixed
by cardiac perfusion with 4% formaldehyde
how was the rat brain visualized in lab
light microscopy
what are the steps for surgery by perfusion fixation
- anesthetize the rat
- make lateral incision through abdominal wall
- incision in diaphragm to expose heart
- clamp sternum and place clamped sternum over the head
- pass perfusion needle through the left ventricle into ascending aorta
- make incision in right atrium to allow blood and perfusate to drain
where is the perfusion needle placed in the heart for cardiac perfusion
through left ventricle into the aorta
right atrium is cut
what is perfused in cardiac perfusion to clear the blood
ice cold saline and sodium nitrate
liver and extremities should be pale
what causes there to be tremors in the rat
perfusion with ice cold formaldehyde in a sodium phosphate buffer
fixative begins to cross link proteins and body becomes rigid
what are rat brains sectioned with after dissection
vibrating microtome and stored in fridge in buffer
how was the rat brain stored in our lab
placed in a 20% sucrose/sodium phosphate buffer at 40 degrees C until it sinks
then frozen in 2-methylbutane at -30 degrees C
what happens if the brain frozen in too hot or too cold of a temperature
too cold: brain will split
too warm: freezing too slow and ice crystals will form (look like swiss cheese)
what is the frozen brain cut with
cryostat or freezing microtome
what are the free floating tissue sections stored in
phosphate budder or cryoprotectant
how do the brain slices stick to the slides
slides are treated to give them a positive charge so that the negatively charged tissue will stick to them
what happens to the sections of brain tissue before they are stained
they are dried
what is cresyl violet
synthetic dye used to stain the cell bodies in nervous tissue purple
what type of staining is cresyl violet staining
nissl staining (stains nissl substance and nissl granules)
what structures are contained in the nissl substance and granules
nissl substance: rough ER
nissl granules: ribosomes
is RNA acidic or basic
acidic and basophillic (bas loving so binds basic dyes like cresyl violet)
what is myelin staining
luxol fast blue used to visualize fiber tracts
what is golgi staining
uses silver nitrate to stain whole neurons, including processes
only stains some, not all neurons
used to quantify the number of dendritic spines under different conditions
what are the basic steps of cresyl violet staining
- submerge in cresyl violet stain
- dehydrate in alcohol baths
- clear tissue with xylenes or histoclear
- coverslip with permount mounting medium
how are the cresyl violet stained sections observed
using bright-field light microscopy
what are the landmarks on the rat skull and how are they created
bregma and lambda
based on the fusion of different skull bone plates
what are the layers of the cerebral cortex
layers 1-6
layer 1 is most external
layer 6 is most internal
what are the two sheets of hippocampus tissue
dentate gyrus (DG) and cornu ammonis (CA)
what are neural circuits in the hippocampus involved in
long term potentiation (LTP)
long term depression (LTD)
what are LTP and LTD important for
learning and memory
what is the pathway of information flow in the hippocampus
- entorhinal cortex to dentate gyrus granule cells via perforant path
- axons of DG (mossy fibers) synapse with CA3 pyramidal cells
- CA3 axons either leave hippocampus via fornix or form schaffer collaterals that synapse with pyramidal cells of CA1
what are the 4 nuclei of the thalamus
ventral posterior
ventral lateral
lateral geniculate nucleus
medial geniculate nucleus
what if the function of the ventral posterior nucleus
somatosensory relay
projects to postcentral gyrus
what if the function of the ventral lateral nucleus
motor
projects to precentral gyrus
what if the function of the lateral geniculate nucleus
visual thalamus
what if the function of the medial geniculate nucleus
auditory thalamus
what are the six nuclei of the hypothalamus
suprachiasmatic nucleus
supraoptic nucleus
paraventricular nucleus
ventromedial nucleus
arcuate nucleus
dorsomedial nucleus
what if the function of the suprachiasmatic nucleus
biological clock
what if the function of the supraoptic nucleus
water balance
what if the function of the paraventricular nucleus
autonomic and neuroendocrine functions
what if the function of the ventromedial nucleus
feeding (satiety signals)
what if the function of the arcuate nucleus
feeding
growth hormone regulation
what if the function of the dorsomedial nucleus
feeding
drinking
circadian activity
what if the function of the olfactory bulbs
sense of smell
what if the function of the caudate putamen
voluntary movement
what if the function of the nucleus accumbens
motivation, aversion, reward, reinforcement behaviors
what if the function of the septum
pleasure center
what if the function of the amygdala nuclei
fear learning and emotional behaviors
what if the function of the piriform cortex
olfaction (3 layer cortex)
what is immunohistochemistry
process of visualizing a specific protein within tissue using an antibody that binds selectively to that protein and is conjugated to something that will enable it to be visualized
what is the equation for pH
pH= -log10([H3O+])
what is a buffer
made when you mix a weak acid with its conjugate base
what are two factors that affect the pH
Ka (equilibrium constant) of the reversible reaction and the relative concentrations of the acid and conjugate base
what is the most common buffer used for immunohistochemistry
phosphate buffered saline
what buffer is used for washing tissue
phosphate buffered saline
what is a blocking buffer used for
blocking non-specific binding of antibody to tissue
what is blocking buffer made of
PBS buffer
bovine serum albumin (BSA)
carrageenan (polysaccharide)
triton X-100
what does triton X-100 do
allows antibodies to pass through cell membranes to bind
what is the pH of buffers used in the lab
7.4
what is the M
mol of solute / L of solution
what are the two types of % solutions
weight in volume (w/v) (g per 100 ml)
volume in volume (v/v) (ml per 100 ml)
what is the difference between the innate immune response and the adaptive immune response
innate: first response to an invading pathogen; quick; non specialized; associated with inflammation
adaptive: slow response the first time a pathogen is encountered but faster for subsequent exposures; very specific; involves T cells and B cells that recognize specific antigens
what is an antigen
anything that generates an adaptive immune response; often proteins but can be polysaccharides or lipids
what is an antibody
glycoprotein that recognizes and binds to a specific antigen
what are antibodies generated by
plasma cells (differentiated B cells) of the adaptive immune system
how many classes of antibodies are there
5
what antibody has the highest concentration in blood
IgG
(generally the one used for immunohistochemistry)
in vivo, what does IgG do
bind to an antigen on an invading pathogen
what is the structure of IgG
4 peptide chains: 2 heavy and 2 light
Y shape
what is at the end of each of the two IgG arms
identical antigen binding site called the Fab region
what is the Fc region
the non specific side of IgG that is similar between different IgG molecules and is glycosylated
what is the difference between a polyclonal and monoclonal antibody
polyclonal: protein is injected into an animal and that animal will generate different plasma cells that produce different antibodies that recognize the different antigens on the protein
- a polyclonal antibody for a particular protein contains multiple antibodies against that protein
monoclonal: comes from cells derived from a single B cell/plasma cell line and each cell makes one type of antibody
- mouse is injected with the protein generating an immune response and that antibody producing plasma cell is fused with a tumor cell to form a hybridoma that will endlessly divide and produce antibody
what is immunohistochemistry
technique of visualizing an antigen in or on cells within a tissue
what are the three main things that immunohistochemistry requires
- tissue that has been processed appropriately
- a monoclonal or polyclonal antibody to bind selectively to the antigen of interest
- a way to visualize the antibody after binding
what are the two ways that there can be visualization of the antibody by light microscopy
- antibody is conjugated to an enzyme that catalyzes a reaction to form an insoluble color product
(peroxidase or alkaline phosphatase) - antibody is conjugated to a fluorophore that can be visualized more directly with fluorescent microscopy (alexa fluors, cyanine dyes)
what kind of fluorescence is used in this lab
direct immunofluorescence
- antibody is conjugated to a fluorophore that can be visualized by exposing it to light of a specific wavelength and detecting the light that is emitted by the fluorophore
what is done to help prevent the non specific binding of the antibody to other proteins and polysaccharides in the tissue
incubate in solution that contains excess of protein (BSA) and polysaccharide (carrageenan)
what are the two antibodies used in the lab
antibody 1 (neurons)
- alexa fluor
- rabbit host
antibody 2 (astrocytes)
- GFAP is the marker
- CY3 fluorophore
- mouse host
what is indirect immunohistochemistry
a secondary antibody that is conjugated to a fluorophore or enzyme is used to detect the primary antibody
what is the ABC method of immunohistochemistry
the avidin-biotin complex method relies on the binding between avidin/streptavidin and biotin
- amplifies the signal because avidin/streptavidin can bind multiple biotin molecules
what are three things about the preparation of an antibody that need to be included for publishing
specific immunizing antigen
species it was raised in
polyclonal or monoclonal
what main control is necessary to show you’re staining what you think you are staining
does the antibody/antiserum stain the tissue of interest from which the molecule of interest has been removed
what are two methods that are used to visualize the brain in post-mortem tissue
in situ hybridization
CLARITY
what is in situ hybridization
used to detect RNA (mRNA) in cells based on DNA-RNA or RNA-RNA hybridization of sense to antisense sequences
- probes are labeled with fluorophore, radioactive, or antigenic tag
when is radioactive labeling useful
when you want to compare the levels of mRNA in cells under different conditions
more mRNA= more radioactive probe hybridized= darker image
what is CLARITY
technique where the post-mortem brain is cleared of lipids, while the proteins and nucleic acids remain
- uses a hydrogel scaffold/mesh to hold the remaining tissue components in place when the lipid is removed
- lipid free cells are permeable to molecular markers like antibodies
what kind of diagram shows the process of fluorescence
jablonski diagram
(shows electronic states)
what are the three stages of a jablonski diagram
excitation
excited-state lifetime
fluorescence emission
what is the excitation stage of a jablonski diagram
photon of energy is supplied by an external source and the energy is absorbed by a fluorophore, creating an excited state
what is the excited-stage lifetime stage of a jablonski diagram
excited state exists for 1-10 nanoseconds and the fluorophore undergoes conformational changes and is subject to a number of different types of molecular interactions
energy is partially dissipated, resulting in a lower energy excited state from which fluorescence emission originates
what is the fluorescence emission stage of a jablonski diagram
remainder of the energy is emitted as a photon of energy returning the fluorophore to its ground state
- during initial energy dissipation the energy of this photon is lower and therefore of longer wavelength than the excitation photon
what is photobleaching
when the fluorophore is repeatedly excited and irreversibly damaged
what is the stokes shift
the distance between the peak excitation and emission wavelengths
- fluorophores with smaller stokes shifts give higher background signal because of the smaller difference between the excitation and emission wavelengths
what is used when there needs to be a distinction between fluorophores
filter cube with a series of 3 filers to only let light within a narrow wavelength range through
draw out light through filter cube
