Immuno Electron Microscopy

IMMUNOGOLD LABELLING TECHNIQUES 

1.         Introduction: The Immunogold labeling technique (IGL) is used to detect surface- and intracellular antigens under electron microscopes, employing antigen-antibody reactions. This technique utilizes electron-dense colloidal gold as a marker/tracer for antigen-antibody reactions and thereby localization of antigens (proteins, hormones, enzymes, neurotransmitters etc.) under a transmission electron microscope. The IGL or immune-electron Microscopy (IEM) may follow two different approaches – (1) immunolabeling before embedding of the cell / tissue samples (called pre-embedding method) and (2) samples are embedded first in resins and then cut sections are immunolabeled (post-embedding method). The standard procedures and reagents that influence the success of IGL in both cases are discussed below: 

1.1.      Antigen presence: If a target antigen is known to be present within the cell, post-embedding IGL gives better results. Similarly, if the target molecule is a surface antigen, pre-embedding IGL is followed. 

1.1.a    Nature of antibodies: Polyclonal antisera comprise a range of antibodies directed against a number of antigenic determinants. Monoclonal antibodies, on the other hand, comprise a single class of antibodies that are directed against a single epitope. Their use becomes apparent when the effects of fixation are considered. Aldehyde fixatives produce strong cross-linking of proteins, which in turn results in conformational changes in the epitopes. These effects hamper the recognition and binding of monoclonal antibodies to tissue epitopes. копии часов

1.1.b   Protein A and Protein G:Several bacterial surface proteins having the property of binding immunoglobulins (IgG) are used in IGL: Protein A and G have been isolated from Staphylococci and Streptococci, respectively and Protein L from Peptococci. Protein A and G have affinity toward IgG and bind to its Fc fragment of many mammalian species. In comparison to Protein A, protein G has higher affinity for IgG molecules from goats, mice and rats, and binds to a wider range of IgG subclasses than does Protein A. In addition, protein G has a good affinity for monoclonal antibodies, a property that is poor for protein A.

1.2  Colloidal gold marker: Since its introduction in IEM by Faulk and Taylor in 1971, colloidal gold has proven to be superior to other markers, such as ferritin. Because of its particulate nature, accurate delineation of the labeled structures in tissues is possible. Being small in sizes (can be < l nm), it allows for the best resolution under TEM. Since it is available in different sizes from 1 to 100 nm, one can perform multiple labeling of various binding sites in the same section. Colloidal gold is a negatively charged hydrophobic sol, formed by electron dense metallic particles. It binds to macromolecules (IgG, Protein A or G) by noncovalent electrostatic stable adsorption.

1.3 Fixative and fixation: Most specimens intended for IGL require fixation for ultrastructural preservation and to render the antigens insoluble. While fixatives maintain cell ultrastructure, they can have a deleterious effect on antigen reactivity. Fixation in high concentrations of glutaraldehyde (>1%) for more than 15 minutes is deleterious to antigens. Lowering the glutaraldehyde concentration (0.1-0.2%) and processing at room temperature with full dehydration of tissue also do not improve the tissue's antigenic response. The tissue is prone to conformational damage from the effects of RT full dehydration and heat polymerization. Minimizing fixation in glutaraldehyde, therefore, necessitates the use of less deleterious dehydration method, such as partial dehydration at RT or progressively lower temperature dehydration to improve the tissue antigenic reactivity. These methods are coupled to the use of special resins (acrylic resins), which can be polymerised at low temperatures.

1.4 Resins: Epoxy resins are widely used for routine TEM studies. However, they are not preferred for IGL, as they form heavy cross-links and exhibit very low water adsorption as they contain many hydrophobic groups in their structures. Both properties inhibit antibody interaction with the antigen in a section. However, the resin can be made hydrophilic by treating sections with oxidising agents such as hydrogen peroxide or periodic acid. The disadvantage of treating sections with oxidising agents is that the alkane group on the antigen is also affected which may reduce the antigenic reactivity. The heavy cross-links formed by epoxy resins are predominantly ester cross-linking and this can be broken by treating the sections with sodium ethoxide. 

1.4.a Acrylic resins:Acrylic resins are more commonly used in IGL because they are more hydrophilic and less densely cross­linked than the epoxy resins, and there is no need for peroxide treatment or etching. The polymerization can be milder than epoxides, at low temperature by ultraviolet rays or at room temperature (RT) using accelerators.  The commonly used acrylic resins are (1) LR white resin (2) LR gold and (3) Lowicryls. 

LR white- The physical and chemical properties of LR white give it versatility as an embedding resin. It is a less viscous liquid capable of penetrating tissues. LR white monomer is partially water miscible and can polymerise in the presence of upto 12% of water, allowing embedding of incompletely dehydrated tissue. LR white resin sections are freely permeable to aqueous solutions. It is available in three grades (i) hard (ii) medium and (iii) soft. The hard grade resin is the one preferred for IGL because it can be considerably underpolymerised and yet retain good sectioning qualities. By reducing the cross-linkage, it is possible to improve the tissue immunoreactivity, by allowing easier access for antibodies. 

Polymerisation of these resins can be achieved by heat, chemical catalysis or UV light. The method chosen will largely depends on the extent of tissue fixation tolerated by the antigen under investigation. Heavily fixed tissues should be heat polymerised. Smaller blocks of delicately preserved tissue better retain their immunoreactivity, if polymerised rapidly with chemical catalysts or UV light in the cold.

LR gold  -  LR Gold is similar to that of LR White. LR Gold has been advocated for embedding "unfixed" tissue at low temperature in order to retain histochemical reactivity of enzymes that are denatured by aldehyde fixation.  Like LR White, LR Gold can be polymerised using UV light at -20°C, but since both resins freeze at -27°C, they cannot be used at the sub -30°C temperature protocols. LR White and LR gold polymerise at room temperature with added accelerators.

Lowicryls  - The most important property of this group of acrylic resins is their low viscosity at temperatures of -350C and below. They can be polymerised with UV light, whilst still at lower temperature. As a result, their most popular applications have been for demonstration of cytochemical reactivity. The lowicryl resins commercially available are Lowicryl K4M (polymerises with UV light at -350C), K11M (polymerises with UV light at -600C) and HM20 and HM23 (polymerise with UV light at -70and -800C, respectively).

1.4.b  Polymerisation:  There are four ways to polymerise resins over a temperature range of 60°C to -80°C:  (i) by heat (ii) chemical catalysts (iii) ultraviolet light  and (iv) blue light.

Acrylic resins can be polymerised by all of the methods listed above, of which heat is the most damaging to the tissue. Chemical, UV and blue-light polymerisation can be carried out at low temperatures (O°C or lower) to retain immunoreactivity.  The cross-linking of acrylic resins is achieved by the presence of free radicals involved in addition reactions on polymerising chains. Free radicals can be produced by heat, in the presence of peroxides or by light. For heat, dibenzoyl peroxide is added as a catalyst for resin polymerisation. Chemical polymerisation at RT or low temperature is again initiated by the addition of dibenzoyl peroxide, but requires an accelerator to produce free radicals, as the activity of the catalyst is linked to temperature. UV light provides the energy to produce free radicals with the help of an initiator (an ethyl or methyl ether).

Ultraviolet light polymerisation: The Lowicryl resins K4M can be polymerised with UV -light at temperature varying between -30°C and -35°C for Lowicryl K4M or as low as -50°C for Lowicryl HM20. An apparatus is made to facilitate indirect UV illumination necessary for even polymerisation of the tissue block. A small, sturdy cardboard box that can be placed in a deep-freezer for low temperature embedding is lined with aluminium foil with shiny side out. A slot is cut in the lid to allow the UV source to be positioned. Two 6 W UV lamps are placed 22-24 cm above the resin filled capsules. A '0' gauge gelatin capsule is used for polymerisation and the capsules should be held in such a way that the resin and tissue receive uninterrupted illumination. The heat produced during exothermic UV polymerisation at RT can result in a brief temperature rise of upto +12°C, if the gelatin capsules are suspended in air but at low temperature, the heat rise is not more than +2°C.

 1.5  Sample preparation for chemically fixed tissues

Cell culture/suspension/free living cells

Cell types can be separated out one from another on the basis of size or weight using density gradients or by simple centrifugation or filtering methods. Cultured cells are removed by scraping them up gently or by using trypsin and collagenase. The cell suspension is centrifuged to a pellet which is immersion fixed directly in Eppendorf tubes. Solid tissue samples are cut into small pieces and fixed.

1.5.a Protocol employing full dehydration of tissue at room temperature: These methods are applicable to large blocks of tissue heavily cross-linked by glutaraldehyde (2-3 mm3) or smaller blocks of post-osmicated tissue (l mm3). All processing steps are completed at RT.

Fixation-  Pieces of tissue are fixed by 1-3% neutral buffered glutaraldehyde for 2-4 hr at RT and then thoroughly washed for 3-4 hr. The tissue blocks are transferred to 50°C ethanol (or acetone) to begin dehydration. Following fixation, treatment of the tissue in 1% ammonium chloride will neutralise residual aldehyde group that can otherwise interfere with immunocytochemical reactivity. 

Protocol  I: For epoxy resinsAll steps are carried out at RT on rotary device unless otherwise stated. A. Ethanol: 1. 50%(15 min.); 2. 70% (2 x 15 min); 3. 90% (15 min); 4. 100% (2 x 30 min). 5.Propylene oxide (2 x 15 min.)

Infiltration; 6. Resin: solvent mixture (1:2) (60 min). 7. Resin: solvent mixture (1:1) (60 min.); 8. Resin: solvent mixture (2:1)          (60 min.); 9. Pure resin (60 min.); 10. Pure resin (Overnight); 11.  Place tissue in resin filled capsules (Polymerisation:  By heat at 600 C)

Protocol 2: This protocol can be used with LR white or Lowicryls K4M and HM20. All operations are carried out at RT on a rotary device unless otherwise stated.    

Dehydration: 50% ethanol(15 min); 1. 70% ethanol (2 x 15 min); 2. 90% ethanol(15 min), 3. 100% ethanol             2 x 15 min 

Infiltration

LR White                                                            Lowicryls

4.     Resin  60 min                                        1:1 resin: ethanol         60 min

5.     Resin    Overnight                                 2:1 resin: ethanol         60 min

6.     Resin      60 min                                        Resin                         60 min

7.     Place tissue in capsules                             Place tissue in capsule 

Polymerisation: For heavily fixed, fully dehydrated tissue heat polymerization is strongly recommended. However, it can also be by any of the other method described earlier. Osmicated samples cannot be UV polymerized.

1.5.b   Protocol employing partial dehydration of tissue at RT and 00C: Sample for partial dehydration should be mildly fixed in glutaraldehyde/paraformaldehyde mixture (0.2% glutaraldehyde + 2% paraformaldehyde). Partial dehydration protocol is only suitable for the more polar acrylic resins able to tolerate at least upto 10% water (i.e. LR White, Lowicryls K4M and K11M).

Protocol 1          RT rapid polymerisation

RT rapid catalytic polymerization methods are devised to avoid the prolonged exposure of tissue to the highly extractive monomeric resin, which is a feature of the heat cure method.

Dehydration: Buffer wash           4 x 30 min; 50% ethanol          1 x 10 min; 70% ethanol         2 x 15 min; Infiltration:  resin             4 x 20 min (at RT)

Polymerization at RT by the chemical catalytic method takes about 30 minutes for LR white or for Lowicryl K4M.

1.5.c  Labeling procedures: This method utilizes labelling with primary antibodies and secondary gold-conjugated antibodies. Gold particles as electron dense markers conjugated with antibodies or protein A and G are most commonly used probe to localize antigenic site in IEM. It has several advantages over other markers (1) unlike other markers  that may fade with time, gold particles give permanent label; (2) because of the particulate nature quantitation is possible even for more than one antigen by multiple labelling with different sized particles.

The size of the gold probe (range 1-100 nm) influences the resolution of labelling. Resolution (distance from gold marker to the site of antigen-antibody interface) is influenced by the selection of size of the gold probe. The best resolution is obtained with a small gold probe complexed antibody

Non-specific labeling (a)   If fixation involves higher concentration of fixative (2.5% glutaraldehyde, 40-60 min.), free aldehyde group must be quenched by NH4Cl (50 nM, 1 hr), otherwise the free aldehyde group will give non-specific labelling. (b) To prevent non-specific binding of antibodies, the specimens are incubated in high ionic strength buffer (buffer   containing upto 500 mM Nacl) or competing inert proteins (blockers). These blockers are dissolved in either phosphate buffer saline (PBS) or tris buffer saline (TBS). The routinely used blockers are ovalbumin, bovine serum albumin (BSA), fish gelatin, defatted milk and acetylated BSA.(c)     Use of Triton X -100 or Tween -20 (0.05%) reduces the non-specific interactions by reducing the charge attraction of antibody.

1.6    Strategies in IGL: The protocol for immunolabeling of resin sections includes pre-treatment, incubation and visualization steps. The pre-treatment procedures that are chosen to optimise the section's immunoreactivity depend on the tissue fixation, the resin employed and the section thickness.All steps are completed at RT on 50-100 nm thick sections adhered preferably on unsupported nickel grids.Epoxy resin sections pre-treatment- Etching for 15 minutes with hydrogen peroxide exposes antigens on the section. Wash with water (1 min).Remove Osmium (if present) - Sodium metaperiodate removes osmium and unmasks some antigen.Inactivate aldehydes (10 min)- Aldehyde groups left in the tissue in fixation cause high background labelling (treat sections in 50 mM NH4cl for 1 hr.).  Blocking Non-specific blocking with large basic protein such as BSA, ovalbumin or gelatin in PBS/TBS for 10 minutes can reduce non-specific background.

 

1.6.a Labeling protocols: Incubation in primary antibody: The diluent may be PBS or TBS, containing BSA, ovalbumin or gelatin. Antibody dilution may be from 1: 10 (monoclonal) to 1: 1000 (for polyclonal antibodies). Incubation of sections overnight at 4°C in diluted primary antibody gives better result than short incubation (1-4 hr) with higher concentration of primary antibody. Wash sections with TBS/BSA (3 x 1 min).

1.6b Colloidal gold conjugate:The secondary antibody conjugated to gold particles should be diluted in TBS/BSA. Incubation in diluted (1:50 or 1:100) antibody for 2-4 hr at RT is the common practice in IGL. Longer incubation, if required, should be carried out at 4°C.

1.6.c  Double labeling procedure: Direct method: As with all other immunocytochemical procedures, double immunolabeling should be preceded by an appropriate section pre-treatment. In the direct method, a cocktail of primary antibodies (two or more), each conjugated to a different sized gold particle, is added in sections for  24 hr. In the indirect method, a mixture of primary antibody A (e.g., a rabbit polyclonal antibody) and primary antibody B (e.g., a mouse monoclonal antibody) is used to incubate sections for 24 hr. Dilution of each antiserum is separately standardised. After wash, a mixture of Anti A conjugated to colloidal gold of a particular size (e.g., 10 nm) and anti B conjugated to colloidal gold of different size (e.g., 20 nm) can be used for incubating the sections for 1 - 24 hr.

1.7   IGL of cell-surface associated antigens: The specimens can be maintained in buffer or lightly fixed and gold labelled prior to dehydration, resin infiltration and polymerisation. Such procedures are generally limited to surface associated antigens in tissue culture or single cell isolates. The method has the advantage that entire surface antigens are available for antibody interaction.

Protocol for pre-embedding IGLFix cells in 0.2% buffered glutaraldehyde for 30 min.Wash in PBS.Wash in 50 mM glycine for 30 min.Incubate for 30 min. at 4°C in PBS containing 1 % BSA, 0.2% sodium azide, and 1.0% normal serum.Wash in PBS containing 0.1% BSA (2x 1 min).Incubate cells in primary antibody diluted in PBS + 0.1% BSA for 30 min at RT.Wash in PBS+BSA.Incubate for 15-60 min. at RT in appropriately diluted secondary antibody-gold complex (diluted in PBS - 0.1 % BSA).Wash, dehydrate and embed in resin.