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immunohistochemistry Flashcards

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1
Q

what are the 2 types of immune response your body has

A

innate immune response

adaptive immune response

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2
Q

what is innate immune response

A

first response to an invading pathogen. very quick and non specialized.

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3
Q

what type of immune response is associated with inflammation

A

innate

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4
Q

what is adaptive immune response

A

slow response the first time a pathogen is encountered. (faster for subsequent exposures) it is ver specific

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5
Q

what types of cells are involved in adaptive immune response

A

t cells and b cells

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6
Q

what are t cells and b cells

A

types of white blood cells/lymphocytes that recognize specific antigens

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7
Q

what are antigens

A

abbreviation for antibody generator. theu are anything that generates an adaptive immune response. they are often proteins but can also be polysaccharides or lipids

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8
Q

what is an antibody

A

a glycoprotein that recognizes and binds to a specific antigen

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9
Q

what generates antibodies

A

plasma cells (differentiated B cells)

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10
Q

what can a plasma cell secrete

A

MANY copies of the SAME antibody that recognizes a specific antigen

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11
Q

how many classes of antibodies are there

A

5

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12
Q

antibodies aka ___

A

immunoglobulin

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13
Q

why is IgG important

A

has the highest concentration in blood and is generally used for immunohistochemistry

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14
Q

structure of IgG

A

made of 4 peptide chains: 2 heavy and 2 light

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15
Q

shape of IgG

A

Y shape with identical antigen beinding site at the end of each of the 2 arms

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16
Q

what is the end of the IgG that binds antigens called

A

Fab region (light chain)

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17
Q

what is the nonspecific end of the IgG called

A

fc region

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18
Q

how is a polyclonal antibody made

A

the protein is injected into an animal and the animal generates diff plasma cells that recognze diff antigens on the protein. blood is drawn from the animal

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19
Q

what is a polyclonal antibody

A

an antibody that contains multiple antibodies against the protein

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20
Q

what is a monoclonal antibody

A

comes from cells derived from a single B-cell/plasma cell line. each plasma cell makes only one type of antibody. only contains a single type of antibody

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21
Q

how is a monoclonal antibody made

A

mouse is injected with the protein which generates an immune response, a specific antibody producing plasma cell is fused with a tumor cell to form a hybridoma. the hybridoma is grown in culture and will endlessly provide and produce antibody

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22
Q

what is immunohistochemistry

A

technique of viualizing an antigen on or on cells within a tissue

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23
Q

ways to visualize antibody by light microscopy

A

antibody conjugated to an enzyme or antibody conjugated to a fluorophore

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24
Q

how does caalyzation of an enzyme to a antibody form a colored product

A

a chemical reaction

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25
common enzymes used
peroxidase and alkaline phosphatase
26
common fluorophores used
alexa flours, dylight flours, flourescein, cyanine dyes, rhodamine
27
what is direct immunofluorescence
antibody is conjugated to a fluorophore that can be viualized by exposing it to light of a specific wavelengtj and detecting light that is emitted
28
how do we decrease nonspecific binding
transfer tissue to blocking buffer at room temp for 1 hour
29
what do we do after soaking the tissue in blocking buffer
incubate the tissue with primary antibodies to detect astrocytes vs neurons
30
what kind of antibody is alexaflour 488
polyclonal
31
what kind of hose is alexaflour 488 from
rabbit
32
what does alexaflour 488 label
neurons
33
what kind of antibody is GFAP
monoclonal
34
what kind of host is GFAP from
mouse
35
what does GFAP label
astrocytes
36
what color is alexaflour 488
green
37
what color is GFAP
yellow/orange. often colored red
38
what is indirect immunohistochemistry
when the primary antibody is conjugated to a second antibody that is conjugated to a fluorophore
39
what does the ABC method stand for and what does it rely on
stands for Avidin-Biotin Complex. it relies on the binding between avidin/streptavadin and biotin
40
what does the ABC method do
amplifies the signal of the fluorophore
41
how does the ABC method amplify the signal
bc avidin/streptavadin can bind multiple biotin molecules
42
what are the methods we can use to tell if the antibody is binding to what we think it is
``` Western blots (good first step) knockout animal (best) antibody shouldnt stain in situ hybridization+immunohistochemistry (hard) similar stainging aptterns as confirmed antibody identical staining pattern to previous description and consistent with classic morphology and distribution ```
43
what is in situ hybridization used to detect
RNA in cells
44
how does in situ hybridization(ISH) work
hybridization of DNA and RNA to RNA. probes labeled with fluorophore, radioactive, or antigenic tag.
45
what is radioactive labeling in ISH used for
to compare the levels of mRNA in cells under diff conditions
46
what is fluorophore labeling in ISH used for
see if 2 mrna's are colocalized in the same cell (tell what type of cell it is)
47
what is CLARITY
brain tissue is cleared of lipids, leaving nucleic acids and proteins. makes brain clear
48
when was clarity developed
2013 by Karl Deisseroth at standford
49
what replaces the lipids in CLARITY
acrylimide
50
point of clarity?
allows you to see a bigger block of tissue
51
what do we do after attaching antibodies to slices
wash sections in PBS to remove unbound antibody
52
what do we use to coverslip the sections in immuno....
vectasheild Hardset mounting medium with DAPI
53
what is DAPI
a fluorescent marker that binds to A-T rich regions of DNA, labeling cell nuclei
54
what is one issue with emission spectra of fluorophores
there is overlap beween the emission of the fluorophores
55
how can we overcome the overlap issue
filter cubes used to only detect a very specific wavelength of light
56
what is the joblinski diagram
shows the three stages in fluors (excitation, excited state, fluorescence emission)
57
how are fluorophores excited
through photon energy of a very specific wavelength
58
whaat is light energy supplied by
mercury vapor lamp, metal halide lamp, or laser
59
what kind of light do we use
metal halide lamp
60
what happens during the excited state of a fluorophore
it undergoes confirmational changes and the energy is partially disipated
61
what does the loss of energy during the excited state cause during the emission state
the energy of the photon emitted is of lower energy and longer wavelength than the excitation photon
62
what is photobleaching
if the fluorophore is excited too much it will be damaged and no longer able to fluoresce
63
how do we limit photobleaching
eposing fluoropore to lowest possible level of excitation for shortest period of time possible.
64
what is Stokes shift
the distance between the peak ecitation and emission wavelengths of a fluorophore
65
what gives a greater background signal: smaller stokes shift or larger stokes shift
smaller
66
what is the excitation filter
the first filter in a filter cube. it only lets through a specific wavelength of light
67
what is the dichromic mirror
the second filter in a filter cube. it is a beam splitting mirror, it further narrows the range of light
68
what is the emission filter
the 3rd filter on the filter cube. it cuts out unwanted fluorescent signals further narrowinf the light range
69
filter cubes: how many ?
one for each fluorophore
70
what color is dapi
blue
71
what does dapi label
cell nuclei

Neuroscience (6 decks)

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