Immunohistochemistry

Question 1

Histological Techniques

Answer 1

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

Question 2

Prep Methods - Fixation

Answer 2

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

Question 3

Embedding

Answer 3

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

Question 4

Dye Staining

Answer 4

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

Question 5

Metal Impregnation

Answer 5

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

Question 6

Immunohistochemistry

Answer 6

Use antibody to detect proteins within tissues
Precise and quantifiable

Question 7

Why do we do tissue prep?

Answer 7

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

Question 8

Tissue Processing

Answer 8

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

Question 9

Sectioning - Microtomes

Answer 9

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

Question 10

Vibratome

Answer 10

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

Question 11

Cryostat

Answer 11

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

Question 12

Sliding Microtome

Answer 12

Frozen samples with CO2 gas or solid CO2
Free floating sections

Question 13

Free Floating Sections

Answer 13

For larger or delicate sections

Question 14

Immunostaining

Answer 14

Uses antibodies against specific tissue or cell

Question 15

Dye Staining

Answer 15

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

Question 16

Nissl Stain

Answer 16

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)

Question 17

Luxol Fast Blue

Answer 17

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

Question 18

Gogli Stain

Answer 18

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

Question 19

Stain Applications

Answer 19

Neuronal injury and diseases
Neurodevelopment - Nissl
Neurodegenerative diseases - Gogli

Question 20

Antigens

Answer 20

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

Question 21

Monoclonal Antibodies

Answer 21

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

Question 22

Polyclonal Antibodies

Answer 22

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

Question 23

Antibody Structure

Answer 23

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

Question 24

Postive & Negative Controls IHC

Answer 24

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

Question 25

Direct Visualization IHC

Answer 25

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

Question 26

Indirect Visualization IHC

Answer 26

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

Question 27

Unmasking

Answer 27

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

Question 28

Blocking

Answer 28

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

Question 29

IHC has 5 steps

Answer 29

Fixation Process Embed Section Stain

Question 30

Antibodies are visualized by

Answer 30

Fluorescence tag Enzyme link (direct/indirect)