Immunohistochemistry

Question 1

What is immunohistochemistry?

Answer 1

Study tissue anatomy and cytoarchitecture

Study distribution of proteins within tissues

Study pathological changes of disease in tissues and cells

Essential for clinical diagnostic neuropathology and basic/translational neuroscience research

Tissue sources: animal models/post-mortem/pathology samples/surplus surgery material

Antibodies raised against a protein (antigen) in a different animal to bind to it and generate a complex - visualised by cutting out enzymatic reaction on a section sample.

Question 2

Why use animal models in immunohistochemistry?

Answer 2

To examine tissue throughout course of disease from early to end stages

Animal models are generated based on specific genetic mutations found in humans

To analyse if potential treatment strategies have any effects

Animal models do not fully replicate human disease so need to interpret results carefully

Welfare of animals must be considered

Question 3

Human post-mortem tissue:

Answer 3

Reduces need to use animals

Better material to study human diseases especially if no animal model exists

End stage study of disease only

Question 4

Autolysis/necrosis:

Answer 4

Once tissue is removed from living beings/post-mortem donor, irreversible autolysis/necrosis occurs = cell damage

Post-mortem tissue = cellular damage more likely

Question 5

Tissue preservation: chemical fixatives:

Answer 5

Chemical fixation:

Formaldehyde = best balance - morphology and staining quality

Chemical fixatives stabilise proteins and macromolecules enmeshed amongst proteins, eg carbohydrates

Cross-linking fixatives, eg Formaldehyde/glutaraldehyde
create covalent bonds between proteins in tissue

Fixation masks/alters epitopes (foreign proteins) to stop them binding to primary antibody

Question 6

Cryopreservation :

Answer 6

Preservation of tissue structure/components by rapid freezing without fixation but can fix tissue and freeze it.

Stops tissue degradation by rapid cooling sample with dry ice/liquid nitrogen

Does not permanently fix tissue - over time degradation will occur rapidly if not stored at minus 80 degrees in freezer.

Snap-freezing = does not change protein structure

BUT: morphology poorly preserved as ice crystals form that damage cell structure.

However, if rapidly cooled to below minus 70 degrees using liquid nitrogen, liquid water converted to vitreous (glass-like) water without crystalline phase so minimises cell damage.

Question 7

Covalent bonds:

Answer 7

Between proteins in tissues lead to good preservation of cell morphology = anchors proteins close to each other inside cells and between cells

Question 8

Precipitating fixatives:
Perfusion:

Answer 8

Ethanol/Methanol

Disrupt hydrophobic bonds between proteins causing irreversible precipitation = create a solid from a solution

Fix tissue by immersing in chemical fixative as a liquid = diffuse through tissue over time

Fixation is slow

Larger samples need stirring (agitation)

Can inject fixative as fluid into circulating system = perfusion - requires in-tact circulation system

Question 9

Perfusion:

Answer 9

Injection of fixative quickly via blood vessels = preferred method

Changes in pH affect reactivity of fixative

Rate of fixative diffusion determines length of incubation in the fixative

Size of specimens = dissected to no larger than 5mm cubes for optimal fixation

Temperature: very important to fixation, eg 4 degrees C = retards degenerative changes but reduces penetration rate of fixative

OR room temperature fixation accelerates fixation penetration but also degenerative changes

Question 10

Embedding:

Answer 10

In solid medium to give extra support to enable tissue sample to be cut into thin sections

Many embedding available: in study of neural architecture, use harder plastic resins so you can cut much thinner

Paraffin wax = most common embedding media and prevents tissue distortion = allows for cutting of different thicknesses

Softer embedding = agarose/gelatine = cut thicker slices

Question 11

Tissue processors:

Answer 11

Dip and Dunk machine = with 12 stations - specimens in basket and stay at stations for set times with agitation (stirring) and automatically transfer from graded alcohols - xylene/Histo-clear - melted paraffin wax before embedding in fresh molten paraffin wax.

Enclosed tissue processor = a chamber in which ingredients pumped in/out under vacuum.

Paraffin wax embedding station = tissues placed in metal moulds filled with molten wax and then placed on cold plate to set the wax.

Question 12

Microtomes and vibratomes:

Answer 12

Plastic cassettes = to enclose tissue - lids removed in embedding - then metal moulds

Sectioning done on microtome - cutting/slicing of samples

Bench-top rotary microtome = used to cut sections from paraffin embedded tissues - thickness ranges from 3-10 microns

Sledge microtome

Paraffin wax sections can be cut as ribbons and floated onto 40 degrees C water to soften wax around samples so samples lay flat so can mount onto slides

Vibratome = used for samples embedded in softer media - NOT paraffin wax but agarose/gelatine instead

Vibratome = 50-100 micron sections can be cut
Sections cut from vibrating blade at high speed but advancing slowly into samples

Stained onto slides/free-floating sections

Cryostat = used for sectioning snap-frozen tissues - cut using refrigerated cabinet at minus 20 degrees C and contains rotating microtome - 8-50 microns

OCT = optimal cutting compound freezes at same density as soft tissues

Sliding microtome = used to cut frozen samples - fitted to bench top, tissue blasted with CO2 gas to keep frozen - 15-200 micron sections

Question 13

Nissl staining:

Answer 13

Dye staining achieved by ionic interaction between dye and tissue

Developed by Frank Nissl - end of 19th Century to identify neurons

Question 14

Luxol Fast Blue:

Answer 14

Acidic dye used to visualise central nervous system myelin axonal sheaths in paraffin wax sections - myelin is stained deep blue

Question 15

Counter stain:

Answer 15

Produces contrasting background colour to main stain

Question 16

Golgi stain:

Answer 16

Metal precipitation (solid formed out of liquid)

Developed by Camillo Golgi (1783)

Modified by Santiago Ramon Y Cajal to draw neurons/circuits - founding study of neuroscience

Small piece of formalin-fixed tissue immersed in potassium chromate then SILVER nitrate

Small random subsets of neurons in exquisite detail - neurons are stained in dark black/brown showing dendritic spines. Colour produced by silver salt deposits in the neuron.

Only some neurons react in Golgi method

Golgi used to study neuronal morphology

Can be used on very thick brain sections up to 500 micrometers or on whole brains.

Used to quantify number of dendritic spines a neuron has - eg loss in schizophrenia

Question 17

Immunofluorescence:

Answer 17

Visualised using fluorescent dyes

Question 18

Immunocytochemistry:

Answer 18

Enzymatic reaction used to visualise antibody antigen complexes on cells grown in laboratory in tissue culture dish

Question 19

Monoclonal and polyclonal antibodies:

Answer 19

Monoclonal = single antibody that recognises specific single epitope (a foreign protein) on antigen molecule

Produced by immunising/injecting animal with antigen (the protein to generate antibody against)

Polyclonal = produced by injecting an antigen of interest into rabbit - rabbit’s B cells will produce antibodies against the antigen - found in serum of rabbit.

A protein has many foreign epitopes and different B cells will make antibodies against different epitopes = polyclonal

Question 20

Antibodies:
Direct/indirect

Answer 20

Y-shaped and have x4 polypeptide chains - belong to the immunoglobulin protein family

x2 heavy chain copies and x2 light chain copies

Y arms contain antigen-binding site

IgG/IgM/IgA/IgD/IgE antibodies

Most antibody reagents are IgG or IgM (added to cause chemical reaction)

To visualise proteins in sections (in membrane of cells) = direct/indirect

Direct = primary antibody applied to tissue sample to bind directly to antigen - only for highly expressed proteins as reporter molecule signal is weak if very few epitopes seen.

Indirect = significantly amplify reporter molecule signal - primary antibody binds to antigen BUT antibody has no reporter linked to it - section incubated with second antibody that binds to primary antibody - second antibody has reporter linked to it.

Secondary antibodies are polyclonal so will react with epitopes all over the primary antibody.

Signal amplified as all secondary antibodies carry reporter molecules.

Reporter molecules are: enzyme/fluorochrome

Fluorescence = antibody (primary/secondary) linked to fluorochrome which is reporter molecule that emits coloured light at wavelength when illuminated by UV light.

Fixation masks/alters epitopes (foreign proteins) to stop them binding to primary antibody

Question 21

Fluorescence microscopes:

Answer 21

View fluorochromes (reporter molecule that emits coloured light at wavelength when illuminated by UV light)

To view multiple proteins using multiple antibodies

Enzymatic detection = antibody linked to enzyme, eg horseradish peroxidase (HRP)

Substrate added to tissue - enzyme and substrate interact to generate insoluble coloured product at site of antibody-antigen complex visualised under light microscope

Substrate = chromogen

Question 22

Positive and negative controls:

Answer 22

Always use positive controls to assess fidelity of technique and primary antibody

Negative control = is making a primary antibody

Immunostaining needs 1) positive/negative controls, 2) antigen retrieval, 3) block non-specific binding

Question 23

HIER: heat-induced epitope retrieval:

Answer 23

Carried out with sections immersed in buffer solution = resists change to pH (acidity/alkalinity)

Question 24

PIER: protein-induced epitope retrieval:

Answer 24

Sections incubated with enzymes, eg Proteinase-K, Trypsin, Pepsin.

False-positive signal = antibodies bind to non-specific components in cells/tissues - low strength

Question 25

Tissue and cell culture:

Answer 25

Cultivation of eukaryotic (cells contain nucleus in organism) tissues or dissociated cells outside of organism in growth media

Cell culture = culturing of dissociated cells

Tissue culture studies cell biology to diagnose chromosomal disorders from blood or amniotic fluid samples, to generate monoclonal antibodies to produce vaccines (Kohler & Milstein, 1975) and IVF (Steptoe & Edward’s, 1977).

Roux, 1885, neural tissue from chick embryos maintained in saline solution for short time.

Question 26

Harrison (1907):

Answer 26

Removed sections of frog embryos, embedded them into blood clots to allow microscope evaluation - he observed growth of nerve cells over many weeks.

Question 27

Burrows & Carrel:

Answer 27

Continued growing cell lines - embryonic chicken hearts.

Question 28

Cell lines:

Answer 28

Can grow indefinitely in culture BUT via genetic mutations and chromosomal abnormalities.

Question 29

Lewis & Lewis (1911):

Answer 29

Grew embryonic chick tissue in liquid media other than blood.

Question 30

Rous & Jones (1916):

Answer 30

Trypsin - dissociate tissues into cells in culture.

Trypsin can cause cell death so now use Accutase or EDTA.

Question 31

Vessels for tissue culture:

Answer 31

Carrel & Baker (1923) = new vessel for tissue culture called ‘Carrel flask’ - angled neck to stop airborne particles.

Modern tissue culture vessels = plastic, sterile, single use plates/flasks/dishes

Dishes = up to 15cm diameter
Plates = 384 wells
Flasks = 25 cm squared - 175 cm squared
Flasks have vented lids to stop airborne particles entering but allow free exchange of gases

Question 32

Biological safety cabinets and tissue culture incubators:

Answer 32

Reduce risk of microbial contamination of cultures

(1909): simple ventilated hood to prepare Tuberculin for mycobacterium tuberculosis = class 1 cabinet

Class 1 = give protection to user/environment from sample BUT do not protect sample from airborne particles.

Class II = most commonly used since 1960’s - rely on continuous uniform flow of clean, filtered air over the sample, air flow splits and flows through grills front and back. Unfiltered air enters and out via filtered exhaust without touching sample.

Class III = hoods - completely enclose sample and have integrated gloves in cabinet

Tissue culture incubators = constant temperature/humidity and levels of gases, eg CO2/oxygen

Question 33

Hayflick & Moorhead (1961):

Answer 33

First cell strains of human fibroblasts W1-38 - they saw primary cells/cell lines/cell strains:

Primary cells = normal tissue and grown without passaging

Cell strains = derived from primary cells

Cell lines = grow indefinitely with abnormal carrier types.

Question 34

Gey (1951):
HeLa cells:

Answer 34

Henrietta Lacks had cervical cancer - Gey generated cell line from a single cell called HeLa cells.

Question 35

Martin Evans (1982):

Answer 35

Isolated embryonic stem cells from mice blastocysts.

Question 36

Jamie Thompson:

Answer 36

Isolated human embryonic stem cells to generate neurons in large numbers.

Question 37

Western Blotting:

Answer 37

To detect specific proteins from mixture of proteins

Transfer proteins in electric field from polyacrylamide gel onto a membrane which blots the proteins from the gel.

Put gel and membrane together in a sandwich between two electrodes.

Sandwich filled out with filter papers/sponges

Membrane made from PVDF/nitrocellulose

Electronic current ran through sandwich and proteins move out of gel and onto membrane

Nitrocellulose and PVDF are very sticky to proteins

Proteins immobilised on membrane and membrane is blocked using milk and add primary antibodies to bind to proteins.

Following adding primary antibody, incubate membrane with secondary antibodies to bind to primary antibody

These secondary antibodies either have:
1) fluorescent tag - gives off light in wavelength
2) have enzyme, HRP (horseradish peroxidase) attached

Add substrate for HRP, enzyme catalyses a chemical reaction that produces light.

Can detect light using photographic film or an imager to pick up signal.

Picture of gel and Western Blot - in blot is only the protein we are interested in due to using specific antibodies.

Question 38

Advantages of Western Blotting:

Answer 38

Takes 1 and a half days - just need glass plates, running and blotting tanks and power supply

Cheap and easy to make - gels and buffers

Works for wide range of proteins and needs a good antibody so all you will see in the gel is your protein you are studying

Can use ANY cell type or tissue - can use to look at changes in protein levels too

However, antibodies often do not bind specifically and pick up proteins not wanted

Sometimes proteins get changed by cell

More difficult if larger proteins or smaller:

Larger = move slowly and do not transfer well

Smaller = run fast and need high % gels

Question 39

Disadvantages of Western Blotting:

Answer 39

Need at least 10% change in protein levels to see on Western Blot

Other techniques, eg QRT-PCR/ELISA more sensitive to measure exactly how much protein

Approximate changes only

Artificial system = cell is making more protein than usually does and the fluorescent tag affects the way the proteins behave at times.