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immunohistochemistry Flashcards

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
Q

common enzymes used

A

peroxidase and alkaline phosphatase

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26
Q

common fluorophores used

A

alexa flours, dylight flours, flourescein, cyanine dyes, rhodamine

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27
Q

what is direct immunofluorescence

A

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

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28
Q

how do we decrease nonspecific binding

A

transfer tissue to blocking buffer at room temp for 1 hour

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29
Q

what do we do after soaking the tissue in blocking buffer

A

incubate the tissue with primary antibodies to detect astrocytes vs neurons

30
Q

what kind of antibody is alexaflour 488

A

polyclonal

31
Q

what kind of hose is alexaflour 488 from

32
Q

what does alexaflour 488 label

33
Q

what kind of antibody is GFAP

A

monoclonal

34
Q

what kind of host is GFAP from

35
Q

what does GFAP label

A

astrocytes

36
Q

what color is alexaflour 488

37
Q

what color is GFAP

A

yellow/orange. often colored red

38
Q

what is indirect immunohistochemistry

A

when the primary antibody is conjugated to a second antibody that is conjugated to a fluorophore

39
Q

what does the ABC method stand for and what does it rely on

A

stands for Avidin-Biotin Complex. it relies on the binding between avidin/streptavadin and biotin

40
Q

what does the ABC method do

A

amplifies the signal of the fluorophore

41
Q

how does the ABC method amplify the signal

A

bc avidin/streptavadin can bind multiple biotin molecules

42
Q

what are the methods we can use to tell if the antibody is binding to what we think it is

A 
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
Q

what is in situ hybridization used to detect

A

RNA in cells

44
Q

how does in situ hybridization(ISH) work

A

hybridization of DNA and RNA to RNA. probes labeled with fluorophore, radioactive, or antigenic tag.

45
Q

what is radioactive labeling in ISH used for

A

to compare the levels of mRNA in cells under diff conditions

46
Q

what is fluorophore labeling in ISH used for

A

see if 2 mrna’s are colocalized in the same cell (tell what type of cell it is)

47
Q

what is CLARITY

A

brain tissue is cleared of lipids, leaving nucleic acids and proteins. makes brain clear

48
Q

when was clarity developed

A

2013 by Karl Deisseroth at standford

49
Q

what replaces the lipids in CLARITY

A

acrylimide

50
Q

point of clarity?

A

allows you to see a bigger block of tissue

51
Q

what do we do after attaching antibodies to slices

A

wash sections in PBS to remove unbound antibody

52
Q

what do we use to coverslip the sections in immuno….

A

vectasheild Hardset mounting medium with DAPI

53
Q

what is DAPI

A

a fluorescent marker that binds to A-T rich regions of DNA, labeling cell nuclei

54
Q

what is one issue with emission spectra of fluorophores

A

there is overlap beween the emission of the fluorophores

55
Q

how can we overcome the overlap issue

A

filter cubes used to only detect a very specific wavelength of light

56
Q

what is the joblinski diagram

A

shows the three stages in fluors (excitation, excited state, fluorescence emission)

57
Q

how are fluorophores excited

A

through photon energy of a very specific wavelength

58
Q

whaat is light energy supplied by

A

mercury vapor lamp, metal halide lamp, or laser

59
Q

what kind of light do we use

A

metal halide lamp

60
Q

what happens during the excited state of a fluorophore

A

it undergoes confirmational changes and the energy is partially disipated

61
Q

what does the loss of energy during the excited state cause during the emission state

A

the energy of the photon emitted is of lower energy and longer wavelength than the excitation photon

62
Q

what is photobleaching

A

if the fluorophore is excited too much it will be damaged and no longer able to fluoresce

63
Q

how do we limit photobleaching

A

eposing fluoropore to lowest possible level of excitation for shortest period of time possible.

64
Q

what is Stokes shift

A

the distance between the peak ecitation and emission wavelengths of a fluorophore

65
Q

what gives a greater background signal: smaller stokes shift or larger stokes shift

66
Q

what is the excitation filter

A

the first filter in a filter cube. it only lets through a specific wavelength of light

67
Q

what is the dichromic mirror

A

the second filter in a filter cube. it is a beam splitting mirror, it further narrows the range of light

68
Q

what is the emission filter

A

the 3rd filter on the filter cube. it cuts out unwanted fluorescent signals further narrowinf the light range

69
Q

filter cubes: how many ?

A

one for each fluorophore

70
Q

what color is dapi

71
Q

what does dapi label

A

cell nuclei

Neuroscience (6 decks)

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