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Histotechnology > Immunohistochemistry and In-Situ Hybridization > Flashcards

Immunohistochemistry and In-Situ Hybridization Flashcards

1
Q

What is immunohistochemistry

A

a staining technique that utilizes antibodies to demonstrate elements of interest in tissues

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2
Q

What is a negative side of IHC

A

it is prone to false-negative and false-positive results and is difficult to troubleshoot

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3
Q

What are antibodies

A

specialized proteins produced by B-lymphocytes in response to antigenic stimulation

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4
Q

How is polyclonal antisera produced

A

the target antigen is isolated and purified then injected into an animal. A mixture of antibodies that bind to different regions of the antigen are formed. They are then eluted from the serum and purified

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5
Q

What is an advantage of polyclonal antisera

A

it is highly sensitive but has low specificity

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6
Q

How is monoclonal antisera produced

A

it begins the same as polyclonal but once the immune response has been mounted the animals spleen is harvested and B-cells are collected. Each B-cell is fused with a non-secreting myeloma cell forming a hybridoma which produces a specific antibody and is immortal

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7
Q

What is an advantage of monoclonal antisera

A

it is highly specific but has low sensitivity

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8
Q

What is a primary antibody

A

an antibody which is specific for the antigen of interest (usually IgG and non-human)

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9
Q

What animal produces murine antibodies

A

mice

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10
Q

What animal produces leporine antibodies

A

rabbits

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11
Q

What are secondary antibodies

A

an antibody which binds the Fc portion of a primary antibody and labeled with a chromogen/fluorochrome to allow visualization

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12
Q

What are tertiary reagents

A

binds to a target on the secondary antibody and carries an activator for the chromogen

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13
Q

What is a chromogen

A

allows visualization of the result

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14
Q

What is the preffered specimen for testing tiny or labile antigens

A

fresh or frozen tissue

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15
Q

What is the preffered specimen for general IHC testing

A

formalin-fixed paraffin-embedde tissue

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16
Q

What are the two classes of epitope retreival

A

heat induced and enzyme induced

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17
Q

What are the advantages of epitope retrieval

A

exposure of previously undetectable antigens
decreased incubation times
ability to use more dilute antisera

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18
Q

What is heat induced epitope retrieval

A

heat and buffered solutions expose antigen sites

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19
Q

What is enzyme induced epitope retrieval

A

proteolytic enzymes digest areas of the tissue exposing antigen sites

20
Q

What is an endogenous peroxidase block

A

primary antibody can bind non-specifically to charged connective tissues. The effect can be neutralized by adding a non-specific protein solution to bind with the charged connective tissues

21
Q

What is a biotin block

A

endogenous biotin can interfere with IHC where biotinylated antibodies are used to detect the binding of the primary antibody. It can be blocked using a two step procedure flooding the slide with avidin to bind biotin and then flooding with biotin to saturate the avidin and then rincing

22
Q

What is direct IHC

A

a labeled primary antibody is applied to the slide and incubated and then the slide is washed

23
Q

What is indirect IHC

A

an unmodified primary antibody is used and then a labelled secondary antibody is applied causing an amplified result

24
Q

What is an immunoperoxidase method

A

enzymes combined with an insoluble chromogen allows a slide to be counterstained using hematoxylin allowing for visualization of IHC and general morphology simultaneously

25
What is a PAP complex
an early form of immunoperoxidase staining, a three step process using a primary antibody an unlabelled secondary antibody and a peroxidase-antiperoxidase immune complex. A chromogen is then added to visualize
26
What is the avidin-biotin method
a primary antibody is followed by a biotinylated secondary antibody then either an unconjugated molecule of avidin is applied followed by peroxidase-conjugated biotin, or a peroxidase-conjugated avidin is used instead
27
What is polymer technology
a polymer is used as a secondary or tertiary reagent in the IHC process. The polymer has many HRP and antibodies embedded within it. It is used in automated IHC instruments
28
What is a reagent negative control
it is treated exactly like the test tissue but the primary antibody is omitted and a diluent is used instead. It is used to identify non-specific staining
29
What is a biological negative control
tissue sections which are known to not contain the antigen of interest it is treated exactly the same as the test tissue. It ensures the stain is negative when it is meant to be
30
What is a positive control
tissue sections which are known to contain the antigen of interest, it is treated the same as test tissue and ensures it is positive when it is meant to be
31
What makes the best positive control
normal tissue that contains the antigen since they tend to express low levels of antigens making then more sensitive controls
32
What causes a test slide and positive control slide to fail
primary antibody not added sunstrate-chromogen mix improperly prepared steps of process not in correct order expired reagents
33
What causes a positive control to be stained well but test sections weakly
low concentration of antigen poorly fixed
33
What causes test slides and positive control to stain weakly
substrate-chromogen mix incorrectly prepared primary antibody too dilute insufficient incubation time too much rinse buffer left on slides diluting reagents insufficient epitope enhancement expired reagents
34
What causes excessive background staining on test and positive control
endogenous peroxidase activity check blocking reagent slides not washed well with buffer between steps tissue allowed to dry during procedure concentration of reagent to high incubation time of primary antibody or chromogen too long excess adhesive
35
What causes excessive background on test but not control
antigen diffusion due to autolysis or necrosis very high levels of endogenous peroxidase pigment in patient slide
36
What causes a negative control to show weak staining
not an issue, it allows us to identify non-specific staining
37
What is in-situ hybridization
similar to IHC but uses a nucleic acid probe instead of a primary antibody and shows a specific sequence of nucleic acids within a tissue sample while retaining morphology
38
What are the benefits of ISH
complementary nucleic acid sequences are highly specific hybridized nucleic acids form a stronger bond provides an alternate means of detection when primary antibodies are unavailable
39
What are the sample conditions for ISH
formalin-fixed paraffin embedded tissue cut 4-6 um and picked up on alcohol-cleaned slides
40
What is the process of ISH
fixed tissue is treated with buffered solutions, heat and proteolytic enzymes to make it more receptive to reagents. DNA is denatured and the probe is applied
41
What is multiplexing
multiple probes used simultaneously to demonstrate multiple targets in the tissue
42
What is a positive control in ISH
contains the target sequence and is treated exactly like the patient slide
43
What is a negative control in ISH
a biological negative
44
What are limitations of ISH
can only been done on fresh or formalin fixed tissue fluorescent methods have usual fluorescent limitations chromogen methods have limited chromogens poorly suited for the detection of single nucleotide polymorphisms

Histotechnology (10 decks)

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