Immunohistochemistry Flashcards
Week Three - 4 lectures
Histological Techniques
Prep tissue/cells + staining + antibodies to identify proteins
Study tissue anatomy, protein distribution, pathological changes
Prep Methods - Fixation
Chemical - formaldehyde, glutaraldehyde
Cryopreservation - rapid freeze (minimize ice crystals)
Embedding
Paraffin wax * (dehydration and solvent xylene)
Plastic resins (thin slices)
Cryoprotective media (OCT provides support)
Dye Staining
Nissl - visualize cell bodies and patterns
Luxol Fast Blue - stains myelin sheaths
Metal Impregnation
Golgi - potassium chromate and silver nitrate
High contrast
For neuronal morphology and visualize entire neuron
Immunohistochemistry
Use antibody to detect proteins within tissues
Precise and quantifiable
Why do we do tissue prep?
Preserve tissue morphology
Fixation affects quality (immersion vs perfusion)
Embedding - sectioning rigidity
Cryopreservation - preserve tissue structure
Tissue Processing
Manual vs Automated
Dip & Dunk (baskets moved through stations)
Enclosed (reagents pumped in vacuum)
Sectioning - Microtomes
Rotary - ribbons in bath then mounted on slides Sledge - hard tissue, precise and consistent
Vibratome
For softer media (agarose, gelatin)
50-500 micron sections
Free float or mounted on slides
Cryostat
Snap frozen tissue -20c
Use vitreous water for embedding or OCT (delicate samples)
Sliding Microtome
Frozen samples with CO2 gas or solid CO2
Free floating sections
Free Floating Sections
For larger or delicate sections
Immunostaining
Uses antibodies against specific tissue or cell
Dye Staining
Selective rather than specific
Use dyes, metals or fluorochromes to induce color contrasts
For living cells in tissue culture
Nissl Stain
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)
Luxol Fast Blue
Acidic dye for myelin sheath
(ex. loss of myelin in neurodegenerative diseases)
Gogli Stain
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
Stain Applications
Neuronal injury and diseases
Neurodevelopment - Nissl
Neurodegenerative diseases - Gogli
Antigens
Recognize harmful threats. Causes body to make immune response against toxins, chemicals, bacteria, viruses
Monoclonal Antibodies
Single epitope on an antigen
Immunization of animal (mouse)
B cells
Grown indefinitely
Secrete specific antibodies
Polyclonal Antibodies
Multiple epitopes on an antigen
Inject antigen into animal (rabbit) and harvesting antibodies in the serum
Antibody Structure
Y-shaped proteins (2 heavy + 2 light chains)
IgG * IgM, IgA, IgD, IgE
Binds at arms of Y (antigen recognition)
Postive & Negative Controls IHC
Positive - confirm fidelity and antibody specs (ex. BrDU in dividing cells)
Negative - ensure any positive is not due to non-specific binding
Direct Visualization IHC
Has marker (uses light - fluorescence or other, labelled on the antibody that binds the antigen)
Indirect Visualization IHC
Secondary antibody is labeled one, and this secondary one reacts with the primary antibody, which in turn binds the antigen
Unmasking
Process of antigen retrieval removes the bonds “masking” the epitope that developed during fixation
Blocking
Agent/buffer that binds to nonspecific protein-binding sites on the membrane so antibodies bind only to their target (ex bovine serum)
IHC has 5 steps
Fixation
Process
Embed
Section
Stain
Antibodies are visualized by
Fluorescence tag
Enzyme link (direct/indirect)
