Modern Immunofluorescence Techniques

Day 1

Overview of Sample Preparation

Or How we go from this…

…to this

First let’s understand what is the purpose of immunostaining

• Use of antibodies to specifically tag intracellular structures

• Antibodies are big molecules impermeable to living cells

• Cells have to be permeabilized but…

– Avoid autolytic processes

– Preserve structure

– Preserve antigenicity

To understand what this means

A brief look of the steps

• Fixation

• Permeabilization

• Blocking

• Antibody incubation

• Counterstaining

• Mounting

• Detection

Fixation

• Is the process of chemical or physical treatment of biological samples aiming to achieve structural and antigenic preservation

• Chemical fixation

– Crosslinkers

– Coagulants

• Physical fixation

– Low temps

– microwaving

Fixation

Chemical fixation

The purpose of chemical fixation is to stabilize the biological sample by either covalent or non-covalent means

Fixation

• Crosslinking agents

– Formaldehyde

– Glutaraldehyde

Fixation

• Both attack nucleophilic groups of proteins:

-NH2, -OH, -SH, =NH

• Formaldehyde is monofunctional and glutaraldehyde is homobifunctional

Fixation

• In the same category we include other chemicals universally designated as bifunctional protein crosslinkers:

– Homobifunctional: e.g., Disuccinimidyl glutarate

– Heterobifunctional:

Fixation Reactivity Class Chemical Group

Carboxyl-to-amine reactive groups Carbodiimide (e.g., EDC)

Amine-reactive groups

NHS ester Imidoester Pentafluorophenyl ester Hydroxymethyl phosphine

Sulfhydryl-reactive groups

Maleimide Haloacetyl (Bromo- or Iodo-) Pyridyldisulfide Thiosulfonate Vinylsulfone

Aldehyde-reactive groups i.e., oxidized sugars (carbonyls)

Hydrazide Alkoxyamine

Photoreactive groups i.e., nonselective, random insertion

Diazirine Aryl Azide

Hydroxyl (nonaqueous)-reactive groups Isocyanate

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Fixation

• How do we prepare a fixative

– We need buffering agent

• Mostly phosphate based buffers or Goode’s buffers (e.g., PIPES, MES)

– We need some salt for ionic strength

• Either KCl or NaCl

– We need some metals for special reasons

• For example Mg2+ and/or Mn2+ cations

– We need chelating agents

• for example EDTA or EGTA

Fixation

An example of fixative solution

MSB (microtubule stabilizing buffer), or PEM (PIPES, EGTA, MgSO4) or BRB (Brinkley’s Buffer)

• 4% w/v PFA ( 0.25% v/v glutaraldehyde)

• 25-80 mM PIPES pH 6.8

• 2.5-10 mM MgSO4

• 5 mM EGTA

We apply fixative for 1 hr to overnight at room temp or in the fridge

Fixation

• Coagulants are typical organic solvents like methanol, ethanol and acetone

• When applied at very low temps (between -80oC to -20oC) they expel water from the hydration sphere of biomolecules causing them to aggregate together

Fixation Pros Cons

Chemical crosslinkers More robust fixation Very toxic

Good preservation of antigenicity* Masking of antigenicity

Excellent preservation of structure* Cumbersome preparation

Slow penetration

Chemical coagulants Fast penetration Poor structural preservation

Good preservation of antigenicity* Might hamper antigenicity

Loss of soluble antigens

Solvents must be anhydrous

Low temps are necessary

Fixation

This is an example of antigen loss following methanol treatment

Fixation

Fixation

Other fixatives

• Non-coagulant:

– Osmium tetroxide

– Potassium dichromate

• Coagulant:

– Zinc salts

– Mercuric chloride

– Picric acid

Fixation

Physical methods

The purpose of physical fixation methods is to vitrify the sample, that is to momentarily immobilize water molecules and biological molecules at the living state avoiding formation of crystalline ice

Fixation

Physical methods

• Plunge freezing

• Cool metal mirror freezing

• High pressure freezing

Fixation

• Plunge freezing

– Tissue is rapidly frozen in liquid ethane or propane

Fixation

• Cool metal mirror slam freezing

The sample is slam frozen on an

LN2 cooled copper mirror

Fixation

• High pressure freeze fixation

The sample is frozen at LN2 temp at extremely high pressure (2000 atm)

Fixation Pros Cons

Plunge freezing Inexpensive Dangerous

Easy to setup Limited sample range

Effective for small samples

Moderate depth of freezing

Cool metal mirror Inexpensive Very limited sample range (monolayers)

Very troublesome optimization (force vs speed)

High pressure freezing

Optimal depth of vitreous ice formation (200 m)

Very expensive

Excellent fixation Needs some optimization

Fixation

• Cryotechniques necessitate embedding to resins and sectioning procedures which make them cost-, time- and labor-demanding

• However preservation of antigenicity and structure is excellent

• Moreover it can be combined with Transmission Electron Microscopy for high quality Correlative Light Electron Microscopy

Permeabilization

• For animal samples the barrier is the plasma membrane

• For plant, fungal and yeast samples the barrier also includes the cell wall

Permeabilization

Plant cell walls comprise of a polysaccharide matrix that is impermeable to macromolecules. Cut-off 20 kDa. Good thing is that polysaccharides are not modified or are minimally modified by chemical fixatives

Permeabilization

• Cell wall digesting enzymes:

– Cutinases

– Cellulases

– Hemicellulases

– Pectinases/pectolyases

– Xylanases

Permeabilization

• Fixed plant material is incubated in cocktails of cell wall digesting enzymes in optimal pH conditions

Permeabilization

The plasma membrane is a lipid bilayer which is also impermeable to macromolecules. Treatment with detergents

Permeabilization

• Detergents are chemicals that form micelles with lipids since they have one polar group that interacts with water and one hydrophobic group that interacts with the lipid

Permeabilization

Ionic detergents (e.g., sodium or lithium dodecyl sulfate (SDS, LiDS), 3-[(3-Cholamidopropyl)dimethylammonio]-1- Propanesulfonate (CHAPS) etc) have a group that becomes ionized at physiological pH. Non-ionic detergents (e.g., Triton series, Tween series, Nonidet etc) contain a polar group which remains non-ionic

Permeabilization

Permeabilization

• Critical micelle concentration (CMC): the lowest concentration of a detergent above which all detergent molecules go into micelles

• Non-ionic detergents have significantly smaller CMC than ionic ones (e.g. CMCSDS=8 mM, CMCTX100=0.2 – 0.9 mM)

• Ionic detergents have strong denaturing effect thus are not preferred

Blocking

• Antibodies interact with antigens quite specifically but they are still proteins so…

– They can be adsorbed to hydrophobic surfaces

– They can associate with polar structures

– They can react with residual aldehyde groups

– If not blocked non-specific binding sites can lead to dirty or erroneous result

Blocking

No blocking Blocking

With the process of blocking we use an irrelevant agent – protein, e.g., BSA – to cover all the sites that can non-specifically adsorb immunoglobulins

Blocking

Sources of artefactual Ig binding

• Fc receptors

• Hydrophobic surfaces

• Charged surfaces

• Epitope mimicry

• Residual aldehyde groups from fixation

Blocking

Blocking

Special cases: Residual aldehydes

• Treatment with glycine

• Treatment with NaBH4 • Treatment with BSA

Blocking

• Glycine and BSA

– The free amino group (-NH2) of glycine or the side amino groups of lysine residues of BSA react with free aldehyde groups

Blocking

• NaBH4

Sodium borohydride is a reducing agent that converts aldehyde residues to alcohols

Blocking

Special case: Fc receptors

Fc receptors are surface transmembrane proteins that bind to the Fc fragment of immunoglobulins irrespectively of their antigenic specificity. Evidently the presence of Fc receptors can lead to erroneous binding of antibodies. Remedy: Saturate Fc receptors with either non-immune serum of the same species or with Fc fragments from a totally unrelated species.

Incubation with antibody

• After extensive blocking, the biological sample is now ready to be incubated with the antibody of choice. When multiple epitopes need to be immunolocalized then one must use immunoglobulins from unrelated animals

Incubation with antibody

Working example

A C B D

F E G H

Counterstaining

Counterstaining is any correlative staining of an immunofluorescent sample that is not based on antibodies

Counterstaining

• Cell cycle determination

This is a metaphase spindle microtubules are stained with mAb against tubulin, kinetochores are stained with pAb against CENP-E and DNA is stained by DAPI

Counterstaining

• Discrimination of cell borders

This can be done with a fixable membrane stain or with a cell wall stain

Counterstaining

Here secondary wall thickenings are stained with the cellulose specific dye calcofluor white

Counterstaining

Mounting medium

• The tissue must be mounted in a medium that:

– Has convenient refractive index

– Contains a strong buffer at optimal pH value for maximum quantum efficiency

– Contains a substance that prevents photobleaching

Mounting medium

• Most common mounting media are mixtures of PBS or TB with glycerol (over 50%) to match or come close to RIs of coverslips, immersion oil and objectives

• We commonly use 90% v/v glycerol in 100 mM Tris-Cl at pH ca. 9.0

Mounting medium

2,2-Thiodiethanol mixtures have very controllable refractive indices

Mounting medium

Fluorescein and relevant dyes such as AlexaFluor 488 have optimum QE at pH>8.0

Mounting medium

Mounting medium

• When the sample is illuminated with high energy excitation light (e.g., UV) free radicals are produced which attack and kill the fluorophore (fluorescence quenching).

• To preserve fluorescence of the sample for adequate imaging we need to include a free radical sequestering agent.

Mounting medium

DABCO: diazabicyclooctane

And other trademarked such as Prolong or SlowFade and so on.

Mounting medium

• p-PDA is by far the best but:

– Very toxic

– Undergoes autocatalytic oxidation

– Slowly soluble to aqueous media

– Needs special care during preparation