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Histology > Immunohistochemistry > Flashcards

Immunohistochemistry Flashcards

1
Q

What is IHC?

A

Locating (known) antigens (proteins) in tissue, using antibodies specific for that antigen.

Antibody is tagged with an enzyme or fluorophore

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2
Q

What is ICC?

A

Locating antigen/protein in a cell or cell compartment using antigen-antibody complex and a biomarker

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3
Q

What is a polyclonal antibody?

A

Multiple different antibodies that bind to multiple epitopes on antigen

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4
Q

What is a monoclonal antibody?

A

1 specific type of antibody binds to 1 specific epitope on antigen

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5
Q

How are monoclonal antibodies made?

A

Mouse injected with antigen.

Lymphocytes isolated (spleen cells)

Spleen cells hybridised with myeloma cells = hybridoma, and rapidly multiply

They are immortalised

Positively selected

Antibody clones selected

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6
Q

How are polyclonal antibodies formed?

A

Multiple antigens injected into mouse(s)

Produces different lymphocytes (Spleen cells) which are isolated and hybridised with myeloma cells

Different types of cells are immortalised and different antibodies are isolated from each cell

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7
Q

Alternative name for antibodies

A

Immunoglobulin

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8
Q

What is the bottom of antibody called?

A

FC fragment - constant domain

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9
Q

What do FC fragments bind to?

A

Cell receptors

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10
Q

What is the top (V) of antibody called

A

FAB fragment - variable domain

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11
Q

What does FAB fragment bind to?

A

Antigen

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12
Q

If indirect IHC is used, what do the primary and secondary antibodies bind to?

A

Primary - Fab fragment binds to antigen

Secondary - Fab fragment binds to primary FC fragment (1st AB = antigen for 2nd AB)

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13
Q

Why is tissue prepared in IHC?

A

Improve antibody penetration

Retrieve antigen (ihc version of dewaxing)

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14
Q

How is tissue prepared for IHC?

A

Heat

Proteolysis

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15
Q

What does heat/proteolysis do to tissue?

A

Reduce cross linking

Makes membrane more permeable
Improves antigen presentation

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16
Q

What is blocking?

A

Blocking unwanted protein binding to the antibody

17
Q

What is used for blocking?

A

Serum of antibody (usually secondary)

18
Q

What is the most common conjugate used for antibodies

A

Horseradish Peroxidase (HRP)

19
Q

Why HRP?

A

Increases detectability of target molecules

Produces weak signal

20
Q

What is the signal produced by HRP in IHC?

What is the other type of signal and it’s substrate name?

A

Insoluble Redish/brown colour

Chemiluminescence with luminol substrate

21
Q

5 steps of indirect detection

A
  1. Blocking reagent - unoccupied membrane sites
  2. Primary antibody specific to antigen - Fab region binds to form complex
  3. Conjugated secondary antibody Fab fragment binds to FC fragment of primary antibody
  4. Substrate addition
  5. ESC = product = signal (colour or chemiluminescence
22
Q

Why do multiple secondary antibody bind to primary antibody FC fragment?

A

FC fragment has repeated amino acid sequences

Or

Secondary antibody in polyclonal

23
Q

What is an alternative conjugate to enzyme/fluorophore?

A

Biotin

24
Q

Which enzyme does biotin interact with?

A

Streptavidin enzyme

25
Q

Where is biotin located in the assay?

A

Secondary antibody in bionylated

26
Q

Why is HRP used in sABC?

A

It cleaves the strepavidin-biotin complex to form the products = signal = light/colour

27
Q

What does streptavidin bind to in IHC?

A

Biotin on secondary antibody

28
Q

When is direct immunoassay performed?

A

Immunocytochemistry

29
Q

Outline direct immunoassay

A
  1. Primary Antibody conjugated with enzyme or fluorophore
  2. Antibody Fab fragment binds to antigen
  3. Substrate added for enzyme or UV light to detect fluorescence
  4. Level of signal proportional to antigen concentration
30
Q

What is direct immunofluorescence?

A

A direct assay - primary antibody conjugated to fluorophore

31
Q

Why does increased signal in IHC/ICC mean?

A

Increased antigen concentration meaning more binding sites for antibody

Histology (5 decks)

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