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KCL Techniques in Neuroscience > Immunohistochemistry > Flashcards

Immunohistochemistry Flashcards

Week Three - 4 lectures

1
Q

Histological Techniques

A

Prep tissue/cells + staining + antibodies to identify proteins
Study tissue anatomy, protein distribution, pathological changes

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2
Q

Prep Methods - Fixation

A

Chemical - formaldehyde, glutaraldehyde
Cryopreservation - rapid freeze (minimize ice crystals)

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3
Q

Embedding

A

Paraffin wax * (dehydration and solvent xylene)
Plastic resins (thin slices)
Cryoprotective media (OCT provides support)

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4
Q

Dye Staining

A

Nissl - visualize cell bodies and patterns
Luxol Fast Blue - stains myelin sheaths

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5
Q

Metal Impregnation

A

Golgi - potassium chromate and silver nitrate
High contrast
For neuronal morphology and visualize entire neuron

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6
Q

Immunohistochemistry

A

Use antibody to detect proteins within tissues
Precise and quantifiable

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7
Q

Why do we do tissue prep?

A

Preserve tissue morphology
Fixation affects quality (immersion vs perfusion)
Embedding - sectioning rigidity
Cryopreservation - preserve tissue structure

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8
Q

Tissue Processing

A

Manual vs Automated
Dip & Dunk (baskets moved through stations)
Enclosed (reagents pumped in vacuum)

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9
Q

Sectioning - Microtomes

A

Rotary - ribbons in bath then mounted on slides Sledge - hard tissue, precise and consistent

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10
Q

Vibratome

A

For softer media (agarose, gelatin)
50-500 micron sections
Free float or mounted on slides

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11
Q

Cryostat

A

Snap frozen tissue -20c
Use vitreous water for embedding or OCT (delicate samples)

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12
Q

Sliding Microtome

A

Frozen samples with CO2 gas or solid CO2
Free floating sections

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13
Q

Free Floating Sections

A

For larger or delicate sections

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14
Q

Immunostaining

A

Uses antibodies against specific tissue or cell

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15
Q

Dye Staining

A

Selective rather than specific
Use dyes, metals or fluorochromes to induce color contrasts
For living cells in tissue culture

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16
Q

Nissl Stain

A

Identifies neurons by staining RNA-rich areas (Rough Endoplasmic Reticulum)
Crystal Violent
For cellular patterns and identify neuron cell bodies
(ex. show loss of hippocampus or abnormal growth)

17
Q

Luxol Fast Blue

A

Acidic dye for myelin sheath
(ex. loss of myelin in neurodegenerative diseases)

18
Q

Gogli Stain

A

Potassium chromate and silver nitrate
Stains random subset of neurons in black/brown
High contrast
Dendritic spines
For neuronal morphology and entire neuron in thick brain sections

19
Q

Stain Applications

A

Neuronal injury and diseases
Neurodevelopment - Nissl
Neurodegenerative diseases - Gogli

20
Q

Antigens

A

Recognize harmful threats. Causes body to make immune response against toxins, chemicals, bacteria, viruses

21
Q

Monoclonal Antibodies

A

Single epitope on an antigen
Immunization of animal (mouse)
B cells
Grown indefinitely
Secrete specific antibodies

22
Q

Polyclonal Antibodies

A

Multiple epitopes on an antigen
Inject antigen into animal (rabbit) and harvesting antibodies in the serum

23
Q

Antibody Structure

A

Y-shaped proteins (2 heavy + 2 light chains)
IgG * IgM, IgA, IgD, IgE
Binds at arms of Y (antigen recognition)

24
Q

Postive & Negative Controls IHC

A

Positive - confirm fidelity and antibody specs (ex. BrDU in dividing cells)
Negative - ensure any positive is not due to non-specific binding

25
Q

Direct Visualization IHC

A

Has marker (uses light - fluorescence or other, labelled on the antibody that binds the antigen)

26
Q

Indirect Visualization IHC

A

Secondary antibody is labeled one, and this secondary one reacts with the primary antibody, which in turn binds the antigen

27
Q

Unmasking

A

Process of antigen retrieval removes the bonds “masking” the epitope that developed during fixation

28
Q

Blocking

A

Agent/buffer that binds to nonspecific protein-binding sites on the membrane so antibodies bind only to their target (ex bovine serum)

29
Q

IHC has 5 steps

A

Fixation
Process
Embed
Section
Stain

30
Q

Antibodies are visualized by

A

Fluorescence tag
Enzyme link (direct/indirect)

KCL Techniques in Neuroscience (4 decks)

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