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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
Q

What is a PAP complex

A

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
Q

What is the avidin-biotin method

A

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
Q

What is polymer technology

A

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
Q

What is a reagent negative control

A

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
Q

What is a biological negative control

A

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
Q

What is a positive control

A

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
Q

What makes the best positive control

A

normal tissue that contains the antigen since they tend to express low levels of antigens making then more sensitive controls

32
Q

What causes a test slide and positive control slide to fail

A

primary antibody not added
sunstrate-chromogen mix improperly prepared
steps of process not in correct order
expired reagents

33
Q

What causes a positive control to be stained well but test sections weakly

A

low concentration of antigen
poorly fixed

33
Q

What causes test slides and positive control to stain weakly

A

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
Q

What causes excessive background staining on test and positive control

A

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
Q

What causes excessive background on test but not control

A

antigen diffusion due to autolysis or necrosis
very high levels of endogenous peroxidase
pigment in patient slide

36
Q

What causes a negative control to show weak staining

A

not an issue, it allows us to identify non-specific staining

37
Q

What is in-situ hybridization

A

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
Q

What are the benefits of ISH

A

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
Q

What are the sample conditions for ISH

A

formalin-fixed paraffin embedded tissue cut 4-6 um and picked up on alcohol-cleaned slides

40
Q

What is the process of ISH

A

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
Q

What is multiplexing

A

multiple probes used simultaneously to demonstrate multiple targets in the tissue

42
Q

What is a positive control in ISH

A

contains the target sequence and is treated exactly like the patient slide

43
Q

What is a negative control in ISH

A

a biological negative

44
Q

What are limitations of ISH

A

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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