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Histo 2 > IHC > Flashcards

IHC Flashcards

1
Q

What is IHC used for?

A

Used to determine the origin of a tumor, prognosis, and treatment

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2
Q

Define: Antibody

A

A host protein (Ig) produced in response to the presence of foreign molecules, organisms, and other agents in the body

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3
Q

How are antibodies produced?

A

Produced by B lymphocytes in response to antigenic stimulation

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4
Q

Define: Antigen

A

A molecule made up of proteins, carbohydrates, or other polymers, and is capable of producing an immune response in animals or cell cultures for the production of antibodies

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5
Q

What are the most common antigens that induce antibody production by the body?

A

bacteria and viruses

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6
Q

Define: Polyclonal Antisera

A

Pool of antibodies created from antigenic stimulation and the production of a mixture of antibodies from many clones of lymphocytes

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7
Q

Why are polyclonal antisera highly sensitive?

A

It binds to multiple epitopes but can cause nonspecific staining

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8
Q

Define: Monoclonal Antibodies

A

Prepared by injecting mice with an antigen
B lymphocytes fuse with non-secreting myeloma cells resulting in hybridomas that retain the antibody secretion capability of the B cell and immortality of the tumor cells

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9
Q

How do you produce unlimited quantities of monoclonal antibodies?

A

By tissue culture or by transplantation into peritoneal cavities of mice

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10
Q

What are the advantages of monoclonal antibodies?

A 
High homogeneity
the absence of nonspecific antibodies
no batch-to-batch or lot-to-lot variability 
purer than polyclonal antibodies 
display the most desirable attributes
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11
Q

What are rabbit monoclonal antibodies?

A

Follows the same principle of monoclonal hybridoma but uses “rabbit fusion partner cells” which fuse to rabbit B cells.
Provide both sensitivity of a rabbit antibody and specificity of targeting a single epitope

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12
Q

Define: Immunofluorescence

A

An insensitive method and antigenic reactivity must be preserved to the maximal extent possible
Makes it possible to visualize antigens in tissue sections or in live cell suspension

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13
Q

What is the classic preparation of tissue for immunofluorescence?

A

Frozen sections of unfixed tissue because antigenic reactivity is minimally impaired and fluorescent antibody staining is strongest

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14
Q

What are the two types of fixation in IHC?

A

Dehydration and Chemical Reagents

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15
Q

Define: Dehydration

A

unfolds and changes the solubility of the protein

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16
Q

Define: Chemical fixation

A

Denatures and stabilizes proteins by coagulation , by forming additive compounds, or by a combination of the 2 actions

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17
Q

What are the 2 categories of IHC Epitope Retrieval?

A

Heat Induced Epitope Retrieval (HIER)
Enzyme Induced Epitope Retrieval (EIER)
Used to break down the hydrogen bonds formed during formalin fixation

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18
Q

What happens if you have overfixation from formaldehyde?

A

It can result in an antibody not having access to it epitope and a false negative

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19
Q

What are the advantages of IHC: Epitope Retrieval?

A

Ability to further dilute antibodies
Exposure of epitope sites not previously detectable
more intense reactions with decreased incubation times
more uniform staining
decreased background staining
day-to-day consistency of stains
improved standardization

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20
Q

Define: HIER

A

Immersing formalin fixed tissue sections in a metallic salt solution and heating in a microwave oven to 100C
pH value of retrieval solution was more important than composition

21
Q

Define: EIER

A

Proteolytic enzyme is used to expose epitope sites
Different proteolytic enzymes may be needed for epitope enhancement of various antigens
It can reduce nonspecific staining but may increase nonspecific staining if not careful

22
Q

What happens if you combine HIER and EIER?

A

combining both methods of epitope retrieval can minimize the extent of enzymatic pretreatment required, HIER is done first, then enzyme digestion

23
Q

How does immunofluorescence work?

A

Reaction sites between antigen and antibody can be visualized easily when a fluorochrome is attached to an antibody

24
Q

What are the most commonly used fluorochromes in immunofluorescence?

A 
Fluorescein isothiocyanate (FITC)
Rhodamine
25
Q

What is the difference between Immunofluorescence and IHC?

A

Frozen kidney and skin biopsy are examined with IF but differentiation of tumors is performed using enzyme IHC

26
Q

Define: Enzyme IHC

A

Enzyme labeled antibodies
The enzyme in the presence of a substrate and a chromogen provides the indicator system to visualize the location of the antibody

27
Q

What enzymes are most commonly used enzymes for Enzyme IHC?

A

Horseradish Peroxidase

Alkaline Phosphatase

28
Q

Define: Direct Method of IHC Staining

A

Moiety bound to primary antibody

a labeled antibody of known specificity is used to identify antigens in the patient’s tissue

29
Q

How is an antibody labeled for Direct IHC?

A

Antibody may be labeled with FITC or other fluorescent dyes for fluorescence microscopy or with an enzyme for subsequent reaction with a chromogen and visualization with the light microscope

30
Q

Define: Indirect Method of IHC Staining

A

Moiety bound to secondary antibody that recognizes primary antibody

Patient’s serum is added to tissue sections containing known antigens to test the patient for the presence of antinuclear antibodies

31
Q

How does the indirect method of IHC staining work?

A

Serum can be added to known bacterium to detect the presence of bacterial antibodies in the patient
A labeled antibody must be used to detect the bound antibodies
Most frequently involve IF microscopy

32
Q

How does Unlabeled or soluble enzyme immune complex method of IHC staining work?

A

3 step method using primary antibody, linking or secondary antibody, and soluble enzyme-antienzyme complex
The complexes must be made in the same animal species for the secondary antibody to link them together

33
Q

How does the avidin-biotin method of IHC staining work?

A

Avidin has a high affinity for the vitamin biotin and binding is essentially irreversible
The primary antibody is followed by a biotinylated secondary antibody (linking antibody)
Third step is the application of a preformed avidin-biotin enzyme complex

34
Q

How does the Polymeric Detection of IHC staining work?

A

dextran polymer and monomer technology
Polymer enzyme molecules such as HRP or alkaline phosphatase have been fused with the secondary antibody
A single strand, allowing for a greater sensitivity and penetration at the antibody binding site
Provides sensitivity without the risk of nonspecific staining when using ABC method

35
Q

What are the staining steps of Polymeric Detection?

A

Antibody
Polymer
Chromogen

36
Q

What are the positive controls of IHC?

A

Must be run with each antibody stain each time it is performed
Optimum control is one prepared under exact same conditions as the diagnostic tissue
Best practice is to use a multi tissue control that contains both positive and negative tissue for an antibody
Control specimens should be placed on the same slide as the patient’s specimen to ensure all steps are the same

37
Q

What are the negative controls of IHC?

A

Run by substituting for the primary antibody or the diluent used for the primary antibody
If diluent buffer is used, the negative control will not detect non specific binding of animal serum components to the tissue and any staining observed will be the result of either endogenous peroxidase activity or binding of other antibody reagents to the specimen

38
Q

What is the first blocking reaction of IHC?

A

The first is the use of hydrogen peroxide to block tissue endogenous peroxidase activity

39
Q

When is the first blocking reaction in IHC essential?

A

If the tissue contains many RBCs

40
Q

What is the second block reaction for IHC?

A

Second block is for nonspecific background staining that may occur a a result of antibody (protein) attachment to highly charged collagen and connective tissue elements

41
Q

How do you prevent non specific binding in blocking reactions in IHC?

A

add an innocuous protein solution to the tissue before the primary antibody is applied
Casein and nondry fat milk are also good blocking agents

42
Q

Protein Expression in IHC

A

Distinguish cells based on expression
IHC - on cultured cells - immunocytochemistry
Use antibodies to recognize specific proteins
Detect antibodies through bound moieties

43
Q

How are proteins visualized in IHC?

A

Colormetric: Visible on light microscope
Fluorescent: more sensitive

44
Q

How are the fluorescent dyes in IHC classified?

A

based on excitation/emission spectra
light absorbed at one wavelength
light emitted at lower energy

45
Q

During an Enzymatic IHC reaction how can you tell if the antibody is there?

A

The reaction gets dark brown

46
Q

Given the following:
Primary: mouse anti-human MHP
Secondary: goat anti-human IgG

what blocking serum would you use? why?

A

Normal goat serum

You dont want the secondary binding all over the place, normal goat serum would block any goat antibodies from binding

47
Q

What does anti-mouse mean?

A

It recognizes and binds any mouse IgG

48
Q

What does Blocking do?

A

it prevents nonspecific binding

Histo 2 (4 decks)

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