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Lab Techniques in Neuroscience > module 3 > Flashcards

module 3 Flashcards

1
Q

what is histology

A

study of microscopic anatomy

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2
Q

what is tissue fixed with for light microscopy

A

4% formaldehyde in phosphate buffer that cross-links proteins so tissue does not degrade

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3
Q

what did we stain rat brain tissue with in lab

A

cresyl violet stain

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4
Q

how was the rat brain in lab fixed

A

by cardiac perfusion with 4% formaldehyde

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5
Q

how was the rat brain visualized in lab

A

light microscopy

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6
Q

what are the steps for surgery by perfusion fixation

A
  1. anesthetize the rat
  2. make lateral incision through abdominal wall
  3. incision in diaphragm to expose heart
  4. clamp sternum and place clamped sternum over the head
  5. pass perfusion needle through the left ventricle into ascending aorta
  6. make incision in right atrium to allow blood and perfusate to drain
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7
Q

where is the perfusion needle placed in the heart for cardiac perfusion

A

through left ventricle into the aorta
right atrium is cut

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8
Q

what is perfused in cardiac perfusion to clear the blood

A

ice cold saline and sodium nitrate

liver and extremities should be pale

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9
Q

what causes there to be tremors in the rat

A

perfusion with ice cold formaldehyde in a sodium phosphate buffer

fixative begins to cross link proteins and body becomes rigid

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10
Q

what are rat brains sectioned with after dissection

A

vibrating microtome and stored in fridge in buffer

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11
Q

how was the rat brain stored in our lab

A

placed in a 20% sucrose/sodium phosphate buffer at 40 degrees C until it sinks

then frozen in 2-methylbutane at -30 degrees C

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12
Q

what happens if the brain frozen in too hot or too cold of a temperature

A

too cold: brain will split
too warm: freezing too slow and ice crystals will form (look like swiss cheese)

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13
Q

what is the frozen brain cut with

A

cryostat or freezing microtome

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14
Q

what are the free floating tissue sections stored in

A

phosphate budder or cryoprotectant

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15
Q

how do the brain slices stick to the slides

A

slides are treated to give them a positive charge so that the negatively charged tissue will stick to them

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16
Q

what happens to the sections of brain tissue before they are stained

A

they are dried

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17
Q

what is cresyl violet

A

synthetic dye used to stain the cell bodies in nervous tissue purple

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18
Q

what type of staining is cresyl violet staining

A

nissl staining (stains nissl substance and nissl granules)

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19
Q

what structures are contained in the nissl substance and granules

A

nissl substance: rough ER
nissl granules: ribosomes

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20
Q

is RNA acidic or basic

A

acidic and basophillic (bas loving so binds basic dyes like cresyl violet)

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21
Q

what is myelin staining

A

luxol fast blue used to visualize fiber tracts

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22
Q

what is golgi staining

A

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

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23
Q

what are the basic steps of cresyl violet staining

A
  1. submerge in cresyl violet stain
  2. dehydrate in alcohol baths
  3. clear tissue with xylenes or histoclear
  4. coverslip with permount mounting medium
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24
Q

how are the cresyl violet stained sections observed

A

using bright-field light microscopy

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25
Q

what are the landmarks on the rat skull and how are they created

A

bregma and lambda

based on the fusion of different skull bone plates

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26
Q

what are the layers of the cerebral cortex

A

layers 1-6

layer 1 is most external
layer 6 is most internal

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27
Q

what are the two sheets of hippocampus tissue

A

dentate gyrus (DG) and cornu ammonis (CA)

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28
Q

what are neural circuits in the hippocampus involved in

A

long term potentiation (LTP)
long term depression (LTD)

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29
Q

what are LTP and LTD important for

A

learning and memory

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30
Q

what is the pathway of information flow in the hippocampus

A
  1. entorhinal cortex to dentate gyrus granule cells via perforant path
  2. axons of DG (mossy fibers) synapse with CA3 pyramidal cells
  3. CA3 axons either leave hippocampus via fornix or form schaffer collaterals that synapse with pyramidal cells of CA1
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31
Q

what are the 4 nuclei of the thalamus

A

ventral posterior
ventral lateral
lateral geniculate nucleus
medial geniculate nucleus

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32
Q

what if the function of the ventral posterior nucleus

A

somatosensory relay
projects to postcentral gyrus

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33
Q

what if the function of the ventral lateral nucleus

A

motor
projects to precentral gyrus

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34
Q

what if the function of the lateral geniculate nucleus

A

visual thalamus

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35
Q

what if the function of the medial geniculate nucleus

A

auditory thalamus

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36
Q

what are the six nuclei of the hypothalamus

A

suprachiasmatic nucleus
supraoptic nucleus
paraventricular nucleus
ventromedial nucleus
arcuate nucleus
dorsomedial nucleus

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37
Q

what if the function of the suprachiasmatic nucleus

A

biological clock

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38
Q

what if the function of the supraoptic nucleus

A

water balance

39
Q

what if the function of the paraventricular nucleus

A

autonomic and neuroendocrine functions

40
Q

what if the function of the ventromedial nucleus

A

feeding (satiety signals)

41
Q

what if the function of the arcuate nucleus

A

feeding
growth hormone regulation

42
Q

what if the function of the dorsomedial nucleus

A

feeding
drinking
circadian activity

43
Q

what if the function of the olfactory bulbs

A

sense of smell

44
Q

what if the function of the caudate putamen

A

voluntary movement

45
Q

what if the function of the nucleus accumbens

A

motivation, aversion, reward, reinforcement behaviors

46
Q

what if the function of the septum

A

pleasure center

47
Q

what if the function of the amygdala nuclei

A

fear learning and emotional behaviors

48
Q

what if the function of the piriform cortex

A

olfaction (3 layer cortex)

49
Q

what is immunohistochemistry

A

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

50
Q

what is the equation for pH

A

pH= -log10([H3O+])

51
Q

what is a buffer

A

made when you mix a weak acid with its conjugate base

52
Q

what are two factors that affect the pH

A

Ka (equilibrium constant) of the reversible reaction and the relative concentrations of the acid and conjugate base

53
Q

what is the most common buffer used for immunohistochemistry

A

phosphate buffered saline

54
Q

what buffer is used for washing tissue

A

phosphate buffered saline

55
Q

what is a blocking buffer used for

A

blocking non-specific binding of antibody to tissue

56
Q

what is blocking buffer made of

A

PBS buffer
bovine serum albumin (BSA)
carrageenan (polysaccharide)
triton X-100

57
Q

what does triton X-100 do

A

allows antibodies to pass through cell membranes to bind

58
Q

what is the pH of buffers used in the lab

A

7.4

59
Q

what is the M

A

mol of solute / L of solution

60
Q

what are the two types of % solutions

A

weight in volume (w/v) (g per 100 ml)
volume in volume (v/v) (ml per 100 ml)

61
Q

what is the difference between the innate immune response and the adaptive immune response

A

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

62
Q

what is an antigen

A

anything that generates an adaptive immune response; often proteins but can be polysaccharides or lipids

63
Q

what is an antibody

A

glycoprotein that recognizes and binds to a specific antigen

64
Q

what are antibodies generated by

A

plasma cells (differentiated B cells) of the adaptive immune system

65
Q

how many classes of antibodies are there

A

5

66
Q

what antibody has the highest concentration in blood

A

IgG
(generally the one used for immunohistochemistry)

67
Q

in vivo, what does IgG do

A

bind to an antigen on an invading pathogen

68
Q

what is the structure of IgG

A

4 peptide chains: 2 heavy and 2 light
Y shape

69
Q

what is at the end of each of the two IgG arms

A

identical antigen binding site called the Fab region

70
Q

what is the Fc region

A

the non specific side of IgG that is similar between different IgG molecules and is glycosylated

71
Q

what is the difference between a polyclonal and monoclonal antibody

A

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

72
Q

what is immunohistochemistry

A

technique of visualizing an antigen in or on cells within a tissue

73
Q

what are the three main things that immunohistochemistry requires

A
  1. tissue that has been processed appropriately
  2. a monoclonal or polyclonal antibody to bind selectively to the antigen of interest
  3. a way to visualize the antibody after binding
74
Q

what are the two ways that there can be visualization of the antibody by light microscopy

A
  1. antibody is conjugated to an enzyme that catalyzes a reaction to form an insoluble color product
    (peroxidase or alkaline phosphatase)
  2. antibody is conjugated to a fluorophore that can be visualized more directly with fluorescent microscopy (alexa fluors, cyanine dyes)
75
Q

what kind of fluorescence is used in this lab

A

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

76
Q

what is done to help prevent the non specific binding of the antibody to other proteins and polysaccharides in the tissue

A

incubate in solution that contains excess of protein (BSA) and polysaccharide (carrageenan)

77
Q

what are the two antibodies used in the lab

A

antibody 1 (neurons)
- alexa fluor
- rabbit host

antibody 2 (astrocytes)
- GFAP is the marker
- CY3 fluorophore
- mouse host

78
Q

what is indirect immunohistochemistry

A

a secondary antibody that is conjugated to a fluorophore or enzyme is used to detect the primary antibody

79
Q

what is the ABC method of immunohistochemistry

A

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

80
Q

what are three things about the preparation of an antibody that need to be included for publishing

A

specific immunizing antigen
species it was raised in
polyclonal or monoclonal

81
Q

what main control is necessary to show you’re staining what you think you are staining

A

does the antibody/antiserum stain the tissue of interest from which the molecule of interest has been removed

82
Q

what are two methods that are used to visualize the brain in post-mortem tissue

A

in situ hybridization
CLARITY

83
Q

what is in situ hybridization

A

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

84
Q

when is radioactive labeling useful

A

when you want to compare the levels of mRNA in cells under different conditions

more mRNA= more radioactive probe hybridized= darker image

85
Q

what is CLARITY

A

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

86
Q

what kind of diagram shows the process of fluorescence

A

jablonski diagram
(shows electronic states)

87
Q

what are the three stages of a jablonski diagram

A

excitation
excited-state lifetime
fluorescence emission

88
Q

what is the excitation stage of a jablonski diagram

A

photon of energy is supplied by an external source and the energy is absorbed by a fluorophore, creating an excited state

89
Q

what is the excited-stage lifetime stage of a jablonski diagram

A

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

90
Q

what is the fluorescence emission stage of a jablonski diagram

A

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

91
Q

what is photobleaching

A

when the fluorophore is repeatedly excited and irreversibly damaged

92
Q

what is the stokes shift

A

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
93
Q

what is used when there needs to be a distinction between fluorophores

A

filter cube with a series of 3 filers to only let light within a narrow wavelength range through

94
Q

draw out light through filter cube

A

Lab Techniques in Neuroscience (3 decks)

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