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Resin (plastic) embedding for microscopy and tissue analysis
Introduction
Resins are used to provide exceptionally strong support in tissue preparation for microscopy. Currently available resins include epoxy and acrylic formulations which can be used in standard histological techniques, but also may be useful in specialized techniques such as immunohistochemistry. The applications, advantages and disadvantages of these agents are described and a comprehensive listing of commercially available resins and resin kits is also included.
The use of resins
Since the introduction of resins into microscopy in the 1950s and 60s ( ), their use has expanded, primarily for electron microscopy. Current techniques and procedures which utilize resins are similar to the basic methods developed in this early period although some methods, and their associated technologies, have been significantly improved, e.g. cryotechniques and microwave-assisted rapid processing and embedding. In addition to their use in electron microscopy, resins are now used in a broad range of techniques and strategies for investigating tissue morphology and tissue components at the molecular level. These include light microscopy, correlative light and electron microscopy, tomography, super-resolution fluorescence microscopy, cytochemistry (histochemistry) and affinity labelling.
Principally, resins are used when there is a need for strong, robust sections which preserve and support the two or three dimensional structure of the tissue and/or the integrity of its molecular constituents. The specific properties of the various resins which make them ideal for this purpose vary depending on the application concerned.
Overall, their general characteristics include many desirable and necessary features:
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Solubility in a range of solvents, e.g. water, alcohol or acetone.
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A range of viscosities are available.
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They polymerize uniformly and in a variety of ways, including at low temperature, but are not usually subject to significant dimensional changes during polymerization.
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Variable hardness but with good plasticity and flexibility so section reasonably easily.
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They lack internal structure and are transparent to light and electrons.
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Some may be etched or removed from tissue sections similar to wax.
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Many are stable under electron bombardment.
In respect to diagnostics and for routine histopathology, commercial paraffin wax and paraffin wax-polymer mixtures are still the most convenient embedding media for the majority of tissues. However, for the methods above, and for some diagnostic applications, resin embedding is superior in some cases and essential in others. Firstly, wax may not give enough support when the tissue and the embedding medium differ significantly in hardness. For example, with un-decalcified bone, especially when the sample is large or dense cortical bone is present, sectioning can be extremely difficult, often producing only fragmented sections. In such cases a more supportive (i.e. hard) resin embedding medium is required which adheres strongly to the specimen, and sectioning may require a range of specialized techniques and equipment, e.g. a motorized microtome. Extremely hard material such as stents (vascular implants) or teeth may only be prepared as slices which are ground down (milled) and polished to the required thickness (a ground section). Again, this requires specialized equipment and procedures such as petrographic techniques which are different from those used for conventional microtomy ( ).
Secondly, wax is too friable for the production of very thin sections for high resolution light microscopy; a problem also related to tissue support, and the hardness, plasticity and flexibility of the embedding medium. For example, whilst high-quality paraffin wax-synthetic resin blends allow sectioning at approximately 2 μm, 1–2 μm resin sections of nerves, renal biopsies and hematopoietic tissues are required to detect minor tissue changes which may be obscured in wax sections.
Thirdly, labile substances such as enzymes may be lost during processing and wax embedding. In these circumstances, resin, rather than paraffin wax may be superior, although some resins, which cannot be removed from the section, may create difficulties with respect to other techniques due to resin masking, e.g. in affinity labelling.
Fourthly, wax is completely unsuitable for electron microscopy as it cannot withstand electron bombardment. In contrast, many resin media (epoxies and some acrylics) have high structural integrity and are relatively stable under electron bombardment, minimizing section damage (volume loss and dimensional changes). For electron microscopy and electron tomography this is an essential property: it is critical that the three-dimensional structure of the cell and the integrity of its molecular components are maintained and only cryosections or extremely thin resin sections (approximately 60-500 nm depending on the application) are suitable.
Note that this chapter focuses principally on the use of resins for light microscopy; the use of resins in electron microscopy is described in Chapter 21 . For the effects of resins on staining, refer to Chapter 9 and techniques for bone are discussed more fully in Chapter 17 . For detailed embedding protocols and additional information on the properties of all resin types see and .
Types of resin embedding media and commercial resin ‘kits’
Three types of resin embedding medium are used in histology and electron microscopy and these are classified according to their chemical composition as acrylic ( Table 8.1 ), epoxy ( Table 8.2 ), or polyester resins. These media generally comprise of one or more resin monomers plus additional components such as a hardener, flexibilizer, plasticizer and accelerator (initiator). Some mixtures may also contain a filler and/or a regulator e.g. Technovit T9100. The various components may be supplied individually or together as a kit. A small number of resins are supplied as ‘pre-mixed’ single solution kits. The polyester resins (Vestopal) are no longer in common use.
Table 8.1
Acrylic resins
Compiled from: www.ScienceServices.eu (accessed December 2017); www.emsdiasum.com (accessed December 2017); www.polysciences.com (accessed December 2017); www.dornandhart.com (accessed December 2017); www.bbisolutions.com (accessed December 2017); www.agarscientific.com (accessed December 2017).
| The following are the principal characteristics and applications of the main commercial acrylic resins and resin kits available from a selection of major histology and electron microscopy laboratory suppliers. Note that there may also be additional proprietary resins available with similar components and specifications. For full technical, processing and application details (including references to key articles), refer to the technical data sheet for the specific resin, or the supplier/manufacturer’s website. |
| Resin | Main techniques | Main characteristics |
|---|---|---|
| Technovit®7100 Glycol methacrylate |
LM 1 μm serial sections Enzyme histochemistry |
Colorless block Polymerization method: C (∼40°C) ± Oxygen sensitive (unsealed embedding) |
| Technovit® 8100 Glycol methacrylate |
LM 1 μm sections Enzyme histochemistry Immunohistochemistry |
Colorless block Polymerization method: C (∼4° and 40°C) |
| Technovit® 9100 Methyl methacrylate |
LM Enzyme histochemistry Immunohistochemistry In situ hybridization Mineralized tissues (heavy duty microtome) Cutting and grinding techniques |
Compatible with most routine LM stains Polymerization method: C (-8 to -20°C) Oxygen sensitive |
| LR White | LM and EM Immunohistochemistry Decalcified bone and teeth Materials science |
Low viscosity (8 cps) Hydrophilic Medium and hard grade Polymerization temperature: 0 to 5°C (exothermic) Polymerization method: C, MW, UV Oxygen sensitive |
| LR Gold | LM 1–4 μm sections and EM Enzyme histochemistry Immunohistochemistry Compatible with unfixed tissue and low temperature techniques (free-floating sections); may not be suitable for dark tissues |
Use polyvinyl pyrrolidone (PVP) to protect unfixed tissue from osmotic damage May be cured at temperatures down to -25°C using a visible light source and benzil Polymerization method: C, L (blue light), UV Oxygen sensitive |
| JB-4 TM Glycol methacrylate |
LM 0.5–2 μm sections High resolution LM Lipid and enzyme retention (cold technique) Calcified bone Embryonic tissues |
Water soluble, complete dehydration through 100% alcohol not required Less shrinkage than wax sections Rapid cure : 90 mins Polymerization method: C (0°C to room temperature, exothermic) |
| JB-4 Plus TM Glycol methacrylate |
LM Similar to JB-4 TM Dense specimens (bone) Temperature-sensitive specimens |
Harder blocks than JB-4 TM Low temperature polymerization; less exothermic than JB-4 TM Polymerization method: C |
| Osteobed Methyl methacrylate |
LM Large and small mineralized (undecalcified) bone specimens Immunohistochemistry |
Large samples: cut with a heavy duty microtome Resin can be removed from the section: staining procedures similar to paraffin sections Polymerization method: C (32–34°C, exothermic) |
| Acrolysin (hard) Methyl methacrylate |
LM 200 μm sections Thick section histology: diamond saws, wires, circular disks and grinders are used for sectioning |
Soft and hard formulations Resin can be removed from the section similar to paraffin sections Polymerization method: C (room temperature: 21–22°C, exothermic) |
| Acrolysin (soft) Methyl methacrylate |
LM 4–10 μm sections Undemineralized bone, hard tissues, and porous bio-materials/implants. Techniques where motorized rotary and sledge/polycut microtomes are used for sectioning Enzyme histochemistry Immunohistochemistry |
Resin can be removed from the section similar to paraffin sections Polymerization method: C (room temperature: 21–22°C, exothermic) |
| Histocryl | LM 1–5 μm sections Routine LM Hard tissues |
Can be sectioned on a steel knife; motorized microtome/glass knife best ‘Water clear’, hydrophilic; most routine LM stains (no etching) Polymerization method: C (exothermic) |
| Lowicryl K4M | LM and EM Temperature-sensitive tissues and techniques, freeze substitution |
Hydrophilic Use down to -35°C Polymerization method: C; UV Oxygen sensitive |
| Lowicryl K11M | LM and EM Temperature-sensitive tissues and techniques, freeze substitution |
Hydrophilic Use down to -60°C Polymerization method: UV Oxygen sensitive |
| Lowicryl HM20 | LM and EM Temperature-sensitive tissues and techniques, freeze substitution |
Hydrophobic Use down to -70°C Polymerization method: C; UV Oxygen sensitive |
| Lowicryl HM23 | LM and EM Temperature-sensitive tissues and techniques, freeze substitution |
Hydrophobic Use down to -80°C Polymerization method: UV Oxygen sensitive |
| Lowicryl Monostep K4M & HM20 |
LM and EM Immunohistochemistry |
Pre-mixed single component kits with the same specifications as the full kits |
| Methyl/butyl methacrylate mixture | LM 1–3 μm sections and EM Normal and mineralized specimens |
Adjustable, soft to hard Resin shrinks during polymerization Can be removed with acetone for LM staining Polymerization method: C; UV |
| Unicryl TM (Bioacryl) | LM 1–3 μm sections and EM Cytochemistry Immunohistochemistry In situ hybridization |
Adjustable, soft to hard Sectioning cleaves block to expose tissue components at surface Resin shrinks ∼10% during polymerization Can be removed with acetone for LM staining Exothermic Polymerization method: C (50–60°C); UV (-10–20°C) |
| Micro-Bed Acrylic-polyester mixture |
LM and EM Immunohistochemistry |
Single component resin, water soluble Hydrophilic Non-cross-linking Similar to wax with most routine LM stains including H&E; polychrome silver-based and cytochemical stains (PAS) also feasible Polymerization method: C (50–60°C); UV (-10–20°C) |
| Immuno-Bed TM Glycol methacrylate based |
LM Immunohistochemistry |
Low viscosity Semi-soluble, may be partially removed Good with routine LM stains; antibodies can penetrate resin Infiltration, embedding and sectioning procedures are similar to JB-4 TM Polymerization method: C (4°C or in a cold room, exothermic) Oxygen sensitive |
| Glycol methacrylate and low-acid glycol methacrylate |
LM 1–3 μm sections and EM Cytochemistry Immunohistochemistry Autoradiography |
Sections of ∼1 μm can be cut with a rotary microtome and steel knife (or glass) and stained with a variety of special stains Thin sections may expand rapidly on water Low-acid version may give improved immunolabelling Water miscible, hydrophilic Polymerization method: UV (4°C or in a cold room, exothermic) Oxygen sensitive (embed in closed gelatin capsules) |
| Glycol methacrylate and polyethylene glycol (PEG) |
LM 1–2 μm sections and EM Cytochemistry Enzyme histochemistry Correlative LM/EM studies (thick and thin sections of the same block) |
Water miscible Alternative formulations with/without the polyethylene glycol Polymerization method: UV Oxygen sensitive (embed in gelatin capsules) |
| 2-hydroxypropylmethacrylate (HPMA) | LM and EM Cytochemistry Soft and hard tissues |
Water soluble: resin acts as dehydrant Water reduces brittleness of blocks May be used with divinyl benzene as a cross-linking agent or PEG to give a lower viscosity mixture (faster penetration, easier polymerization) Polymerization method: UV (3°C); C (60°C) Oxygen sensitive (embed in gelatin capsules) |
cps: centipoise; EM: electron microscopy; LM: light microscopy
Polymerization method: chemical accelerator (may also require heat) (C); light (L); ultraviolet light (UV); microwave oven (MW)
Table 8.2
Epoxy resins
Compiled from: www.ScienceServices.eu (accessed December 2017); www.emsdiasum.com (accessed December 2017); http://www.taab.co.uk/pdf-products.php?cat_id=2&sub_cat_id=19&pdf_id=6 (accessed December 2017); http://www.polysciences.com/default/catalog-products/life-sciences/ (accessed December 2017).
| The following are the principal characteristics and applications of the main commercial epoxy resins and resin kits available from a selection of major histology and electron microscopy laboratory suppliers. Note that there may also be additional proprietary resins available with similar components and specifications. For full technical, processing and application details (including references to key articles), refer to the technical data sheet for the specific resin, or the supplier/manufacturer’s website. |
| Resin | Main techniques | Main characteristics |
|---|---|---|
| Epo-Fix | LM Materials science and metallographics Hard samples & complicated shapes Vacuum embedding |
Low viscosity Good specimen adherence Polymerization method: C (RT 8 hrs; 60°C 2 hrs) |
| Epon 812 (Embed 812, Ladd LX112) Embed 812/DER 736: low viscosity version of Embed 812 |
EM (LM) | Polymerization method: C |
| Hard-Plus resin 812 Modification of Epon 812 |
EM (LM) | Lower viscosity than Epon 812 Intermediate solvent not required for embedding Polymerization method: C |
| Araldite CY212 ( Araldite M ) Diglycidyl ether of bisphenol A |
EM (LM) | Viscosity: 1300-1650 cps (25°C) Polymerization method: C |
| Araldite 502 | EM (LM) | Viscosity: 3000 cps (25°C) Polymerization method: C |
| Araldite 6005 ( GY 6005 ) American Araldite |
EM (LM) Hard specimens which do not require the resin to infiltrate the specimen (slow penetration) |
Penetrates slowly Blocks slightly harder than Araldite 502 Polymerization method: C |
| Araldite/Embed 812 ( Epon 812 ) | EM (LM) | Hard blocks/high image contrast Blocks easily sectioned Polymerization method: C |
| DER 332/732 TM Liquid reaction product of epichlorohydrin and bisphenol A |
EM (LM) | 3 mixtures: soft block/hard block (collagenous tissue)/very hard Polymerization method: C |
| Quetol 651 Ethylene glycol diglycidyl ether |
EM (LM) Immunohistochemistry |
Water miscible, acts as a dehydrant Water reduces cross-linking Polymerization method: C |
| Durcupan ACM Aromatic polyepoxide Variation of Araldite casting resin M |
EM (LM) Alternative to methacrylate |
Low viscosity, low shrinkage Polymerization method: C |
| Durcupan Aliphatic polyepoxide |
EM (LM) | Water soluble Polymerization method: C |
| UltraBed Modified Spurr’s formulation |
EM (LM) | Low viscosity: 65 cps (25°C) Polymerization method: C |
| ERL 4221 (Spurr’s) Replacement for VCD (ERL 4206) |
EM (LM) | Low viscosity Hardness adjusted using flexibilizer DER 736 Polymerization method: C |
cps: centipoise; EM: electron microscopy (brackets indicate the resin is not specified for this use, but may be suitable); LM: light microscopy; RT: room temperature
Polymerization method: chemical accelerator (may also require heat) (C)
Prior to use, the resin monomers and other components are mixed together in specific proportions, the ratios being changed to alter the physical and chemical properties of the liquid resin mixture, the mode of polymerization (i.e. the change from liquid resin mixture to solid block), and the physical and chemical properties of the final polymerized block. Importantly, when using multicomponent resin systems, it is critical to ensure that all the components are accurately measured and thoroughly mixed in order to produce good quality blocks with a consistent internal composition. It should be noted however, that the infiltration rate of each resin component will depend on tissue density and the size of the diffusing molecules and that, after mixing, when the polymerization process has been activated, the resin may start to increase in overall viscosity. Longer infiltration times are required for more viscous resins. It follows that, if tissue infiltration is incomplete for any reason, polymerization may not be uniform.
Depending on the resin involved, and the imaging and labelling techniques to be applied, polymerization may be carried out chemically, or by using heat (a standard or microwave oven) or light (UV or blue light). Resins which cure with an exothermic reaction may need to be cooled or polymerized in the cold (approximately 4°C or less). Oxygen-sensitive resins, mainly acrylics, must be polymerized in a vacuum, in an inert gas atmosphere e.g. dry nitrogen or in sealed embedding molds which are impermeable to atmospheric oxygen. In addition, low viscosity oxygen-sensitive resins should not be mixed vigorously, or for long periods, to avoid drawing oxygen into the resin.
A wide range of resins are now available from the major histology and electron microscopy laboratory suppliers. The diversity of commercial products reflects improvements in resin technology and an increase in the range of applications which utilize resins. While this means that there are now resins designed for particular techniques, the diversity of products (especially acrylics) can make it difficult to identify the best resin and/or formulation for any specific application. The problem is exacerbated by the fact that several proprietary kits may claim to be suitable for the same application because some formulations are marketed under different names (especially the Technovit range) ( ). Furthermore, in some cases the original resin monomers described in the literature may no longer be available and commercial kits contain substitutes (e.g. Epon 812 and Spurr resin ERL 4201 – see ).
Resin formulations may be designed principally for either light or electron microscopy, or both. The latter (e.g. LR White and Lowicryl K4M) facilitates combined light and electron microscopy examination of the same block or, in correlative microscopy, the same section ( ). Epoxy resins are favored for routine electron microscopy as they cross-link with the specimen, provide excellent ultrastructural preservation and are stable in the electron beam.
Acrylics are preferred for light microscopy, although many can also be used for electron microscopy and for cytochemical and affinity labelling techniques. The acrylic resins best suited to cytochemical and affinity labelling are likely to be those which can be polymerized at low temperature (or with a low polymerization exotherm), thus minimizing protein degradation and damage to cellular structures. In this respect, some acrylics are compatible with vitrified specimens and freeze substitution techniques, properties also desirable for correlative studies and super-resolution fluorescence microscopy ( ). Resins which do not cross-link with the specimen or require alcohol dehydration are also useful for cytochemical and affinity labelling techniques, as these too can damage cellular components and hinder tissue-probe interactions. Some acrylic and epoxy resins can be easily etched or removed from the section, thus reducing tissue masking and improving access to cellular components.
When resin media were first introduced, knowledge of the chemistry and interactions of the various components was critical for optimizing results. Commercial resins now come with recommendations on their formulation and use for specific applications, as well as details of their chemical and physical characteristics. Some formulations can also be easily modified to change the hardness of the final block. Note that many resin components are toxic and/or hazardous and present potential health and safety problems (particularly contact dermatitis). It is important that all the chemicals used in resin media are handled in accordance with local workplace safety regulations.
Cryotechniques
Cryotechniques are used with fixed and unfixed tissues to immobilize tissue components and improve cell preservation especially for tomography, super-resolution fluorescence microscopy, cytochemistry and affinity labelling studies. Epoxy and acrylic resins can both be used as freeze substitution media. Schedules for epoxy resins and acrylics (LR White, Unicryl and the Lowicryl series) are given by ; methods for epoxy resin (Epon 812/Araldite 506) and the Lowicryl series are given by . A method for correlative super resolution fluorescence and electron microscopy using cryo-fixed samples embedded in Lowicryl HM20 is described by .
Super resolution fluorescence microscopy, correlative microscopy and tomography
Super resolution microscopy enables the visualization of single molecules through fluorescent imaging. For reviews, see: . For this application, used high pressure frozen tissue which was freeze-substituted and embedded in LR White at -20°C. As the shelf-life of the resin with added catalyst for use at this temperature is reduced to about one month, the resin is normally stored without the catalyst which is added just prior to use ( ). Sections are cut at 100 nm. For a correlative study, used Lowicryl HM20 monostep resin to produce sections which were each examined by both super resolution and electron microscopy.
Transmission electron tomography (TEM tomography) is used to investigate the 3D architecture of the cell in sections 200–500 nm thick. A focused ion beam scanning electron microscope (FIB-SEM) is used to image larger volumes ( ). The stability of the sample and embedding medium, i.e. resistance to mass loss and 3D shrinkage, is extremely important in this technique and the choice of resin is critical. In a comparative study of standard and modified formulations of epoxy resins (Epon, Durcupan, Epon/Durcupan mixtures and Hard Plus 812) and methacrylates (Lowicryl HM20, K11M, HM20/K11M mixtures), found that Hard Plus with 10% linear shrinkage and 15% mass loss was the resin of choice. For immunolabelling studies and correlative fluorescence light microscopy, Lowicryl K11M or K11M/HM20 were a good alternative.
Rapid embedding
Rapid resin embedding and polymerization are extremely useful for the speedy turnaround of diagnostic material and to reduce the deleterious effects of liquid resin, e.g. lipid extraction on tissue samples ( ). General schedules for epoxy and acrylic resins are described by and . describes microwave-assisted techniques. The embedment of frozen tissue into epoxy and acrylic resins using centrifugation for rapid resin infiltration, followed by high temperature polymerization, is described by . One should note that this novel technique may possibly be adapted for routine samples.
Sectioning resin-embedded material
The majority of resin media require the use of a motorized microtome and specially designed blades rather than standard disposable steel blades in order to cut good quality sections (see also Chapter 7, Chapter 17 ). The complete range of knives, varying by composition, coating, size and profile available for resin histology and their specific applications is too extensive to be fully discussed here. Full details can be found on the various manufacturer and supplier websites. Briefly, tungsten carbide knives are suitable for acrylic resin applications, including bone, hard materials, stents and for frozen sectioning. Some metal knives are coated with a material such as amorphous diamond, teflon, polymer, ceramic or titanium nitride to reduce friction and increase the longevity of the cutting edge. Diamond-coated metal knives are designed specifically to cut hard materials and various sizes are now made for both light and electron microscopy. Sapphire knives are a cheaper alternative. However, the knife edge of both diamond and sapphire is fragile and easily damaged and, while both will cut resin blocks, neither are suitable for hard material or tissue with hard inclusions. Most resins can be cut on an ultramicrotome with a triangular Hartmann-Latta glass knife (as used in electron microscopy) providing the block is not too large. Rectangular Ralph glass knives, with a long cutting edge are used for standard microtomy. All glass knives are quickly damaged by hard material or inclusions.
Acrylic resins
The acrylic resins used for microscopy are esters of acrylic acid (CH 2 ··CH·COOH) or, more commonly, methacrylic acid (CH··C(CH 3 ) ·COOH) and are commonly referred to as acrylates and methacrylates respectively. Some acrylics are water miscible and can be used without the use of a dehydrating solvent. Numerous formulations have been devised with a wide range of properties and applications ( Table 8.1 ). Conventional acrylics are polymerized (cured) by complex free-radical chain reactions, the radicals usually being produced from the breakdown of a catalyst, most commonly benzoyl peroxide. Radicals can also be produced spontaneously by light or heat so acrylic resins should be stored in dark bottles in a cool place.
Benzoyl peroxide breaks down at 50–60°C but the addition of a tertiary aromatic amine (such as N , N -dimethylaniline or dimethyl p -toluidine) causes it to produce radicals at 0°C and this allows polymerization at low, or room temperature. Dry benzoyl peroxide is explosive and is supplied damped with water as a paste mixed with dibutyl phthalate, or as plasticized particles. For use in some formulations the water must be removed and care must be taken to dry aliquots away from direct sunlight or heat. Light-sensitive photocatalysts such as benzil and benzoin are used for polymerization of acrylics at sub-zero temperatures using short wavelength light.
In addition to the monomer and catalyst, several other ingredients are often necessary in acrylic resin formulations. Amines cause polymerization to proceed at a faster rate and are termed as either accelerators or, more rarely, as activators. Other accelerators include sulfinic acid and some barbiturates. To improve the sectioning qualities of acrylic blocks, softeners or plasticizers are often added to the mix. Typical examples are 2-butoxyethanol, 2-isopropoxyethanol, polyethylene glycol 200/400 and dibutyl phthalate. Some acrylic mixes need a small amount of a cross-linker such as ethylene glycol dimethacrylate to stabilize the resin and protect it from the physical damage which may otherwise be caused by an electron beam, e.g. Lowicryl resins, or staining solutions, e.g. Technovit 8100. Unlike epoxy resins, the viscosity of acrylics is low and whilst relatively short infiltration times are possible, the size and nature of the tissue, together with the processing and embedding temperature, will affect the length of incubation required.
2-hydroxyethyl methacrylate (HEMA), more commonly known as glycol methacrylate (GMA), is a popular embedding medium for light microscopy since it is extremely hydrophilic and allows the use of a wide range of tinctorial staining methods. GMA also sections well providing the block is kept dry. Various mixes have been described and commercial kits are available. Most mixtures are based on the formulation published by . Although the mixes all contain GMA, the proportion and variety of the monomer and other ingredients may vary; this means that, comparatively, commercial kits may have different characteristics. The monomer can also be contaminated with methacrylic acid which may result in background staining. This can be reduced by purchasing low-acid GMA or a high-quality proprietary kit such as JB-4, JB-4 Plus (Polysciences Inc., USA), Technovit 7100, or Technovit 8100 (Kulzer, Germany). Butyl methacrylate is now rarely used as the main monomer in any histological formulation as it is unreliable and produces considerable tissue artifact during polymerization. However, butyl methacrylate can be added to some acrylic formulations, e.g. Unicryl (BBI Solutions, Wales, UK) and can be used to modify the hardness of methyl methacrylate blocks.
Some formulations are based on aromatic polyhydroxy dimethacrylate resins (Histocryl, LR White & LR Gold from London Resin Company, UK: see Table 8.1 ). Histocryl is intended for light microscopy but LR White and LR Gold can be used both for light and electron microscopy as they are hydrophilic and stable in an electron beam. LR White may be polymerized by the addition of dimethyl p -toluidine; LR Gold is cured by the addition of benzil and exposure to light from a quartz halogen lamp for low temperature embedding. Other acrylic resins cured at low temperature include the Lowicryl range and Unicryl. The Lowicryl range may also be cured using the photocatalyst benzoin and ultraviolet light. Lowicryl K4M Plus is a light-curable epoxy-acrylate product combining rapid polymerization of the acrylic component with the high strength of an epoxy.
For many years methyl methacrylate (MMA) has been widely used in microscopy because its hardness makes it an ideal embedding medium for undecalcified bone, other hard tissues and tissues with stents or implants. Proprietary kits for these applications include Technovit 9100 (Kulzer), OsteoBed (Polysciences) and Acrylosin (Dorn & Hart Microedge Inc.). Variations of the MMA formulation allow tinctorial staining and immunolabelling of semi-thin sections for high-resolution light microscopy. Disadvantages of MMA are that it may polymerize with considerable shrinkage and it is a powerful lipid solvent, even at low temperature ( ).
Applications and characteristics
Acrylic resins can be used for both light and electron microscopy and are therefore useful for correlative light and electron microscopy. Some, such as the Lowicryl range, have been developed mainly for electron microscopy ( ) but LR White and Unicryl ( ) can be used for either. For various technical reasons, not all dual-purpose resins are practical for routine high resolution light microscopy studies.
Hydrophilic resins such as GMA and LR White allow tissue to be stained without removal of the embedding medium and are popular for routine use. Many ‘simple’ staining techniques may be applied, but some may require modification of the standard method for paraffin sections. All acrylic hydrophilic media are insoluble, consequently all staining occurs with the resin in situ. This can cause two problems: either the medium itself becomes stained, or the matrix acts as a physical barrier, masking the tissue. As noted earlier, acrylic resins may be polymerized using a chemical accelerator, heat, or light and an advantage of some acrylics is that they can be polymerized at low temperature (down to -80°C). The optimal polymerization method will depend on factors such as the imaging, staining and labelling techniques required, the technical practicality of the method, and the equipment available (particularly in respect to low temperature techniques and oxygen-sensitive formulations). Note that resins with an exothermic polymerization reaction may need to be cooled or polymerized in the cold (approximately 4°C or less).
Tinctorial staining
Acrylic sections stain easily and are therefore popular for high-resolution light microscopy; excellent results can be achieved with GMA formulations as well as resins such as LR White, even though the resin cannot be removed. Numerous, but not all, routine histological staining methods for paraffin sections may be applied to sections, e.g. H&E, PAS, van Gieson, alcian blue, Perls’, elastic methods, Giemsa, and silver techniques for reticulin, although some methods may need to be modified. For example, media in the London Resin range (Histocryl, LR White, and LR Gold) are all softened by alcohol; this means that alcoholic staining solutions such as those used in elastic methods can easily result in section loss. Consequently, even hematoxylin staining of a London Resin should be progressive to avoid exposure to acid alcohol during differentiation; regressive staining of GMA sections is possible with care. The embedding medium, especially GMA, may also stain, although in some techniques this can be reduced by careful washing. Penetration of stains into GMA and the level of background staining and its removal, depend on the molecular size of the dye and the level of resin cross-linking. Dyes with large molecules (MW approximately 1000) penetrate and are lost slowly; small dyes (MW < 550) penetrate, and are lost quickly. Increased cross-linking inhibits stain penetration ( ). The hydrophilic/lipophilic character of the staining reagent also affects the level of background; lipophilic stains producing intense background staining ( ). A numerical guide which helps to avoid artifacts resulting from hydrophobic and size effects is given by .
In contrast to GMA, MMA can easily be removed prior to staining using similar procedures and solutions to those used routinely for dewaxing paraffin sections, but with slightly extended incubation times. Tinctorial staining of tissue in MMA is possible without removing the resin if no cross-linker has been added; this can be useful for sections of undecalcified bone prepared as described in Chapter 17 (except for semi-thin sections for high-resolution light microscopy).
Enzyme histochemistry
The ability to process, embed and polymerize some acrylic resins at low temperature allows a variety of enzymes to be demonstrated in tissue sections. The use of GMA for enzyme histochemical studies is popular as this resin is easy to handle and produces good results. has shown that enzyme activity may be affected during each step of the processing schedule thus, the effects of each step (and the reagents involved) should be tested on the enzyme of interest in order to maximize staining. Although some enzymes are destroyed by routine fixation and processing, a large number have been successfully demonstrated. A simple protocol is to carry out fixation, processing and polymerization at 4°C and then dry the sections onto a coverglass (or slide) at room temperature overnight prior to performing enzyme histochemical staining. After staining, care must be taken to ensure that enzyme diffusion and loss do not occur during the washing and mounting procedures.
A variety of aldehyde fixatives have been advocated for histochemical studies but 10% formal calcium is recommended ( ). Staining may be enhanced if the tissue is subsequently washed at 4°C in 3% buffered sucrose solution. Polymerization is normally carried out at 4°C using a chemical accelerator, but for methods utilizing sub-zero temperatures either an excess of catalyst, or a photocatalyst, can be employed, depending on the resin formulation.
In some cases it may be necessary to avoid fixation. describe a method for processing fresh (unfixed) tissue stabilized with polyvinyl pyrrolidone (MW 44,000) at -25°C and embedded in LR Gold. Polymerization was achieved using the photocatalyst benzil and blue light from a quartz halogen lamp. This procedure has the potential to demonstrate fixation-sensitive enzymes such as the oxidative enzyme succinate dehydrogenase.
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