We recommend bubbling the MonoSteps for 5 minutes with nitrogen gas before use to mix them well and to replace solved gases, which can impede polymerization.
Initially, low-temperature embedding with LOWICRYL resins was developed to improve the preservation of the cellular ultrastructure [Kellenberger et al., 1980; Carlemalm et al., 1982], and to image unstained sections of biological material in the scanning transmission electron microscope (STEM) [Carlemalm and Kellenberger, 1982]. Depending on the actual composition of the LOWICRYL resins, the two basic types K4M (polar) and HM20 (nonpolar) may be used at temperatures down to -35°C (K4M) or -50°C (HM20).
Exploring cryofixation and freeze-substitution led to the development of two additional LOWICRYL resins, the polar K11M, and the nonpolar HM23 [Carlemalm et al., 1985; Acetarin et al., 1986]. The latter two resins may be used at temperatures down to -60°C (K11M) or -80°C (HM23). It was demonstrated quite early that low-temperature embedding with the polar LOWICRYL resin (K4M also let to significantly better preservation of organelle structure and antigenicity [Rothe et al., 1981] compared to that obtained after classic fixation and embedding protocols.
Both polar and nonpolar LOWICRYL resins are highly suitable for immunolabeling and lectin-labeling [see e.g. Durrenberger et al, 1991; Roth, 1989], thus explaining their frequent use in immunocytochemistry both at the light and electron microscope level.
The Lowicryl series of resins is mixed from three components by the user.
The new MonoStep resin (polar and nonpolar) were the successors of these resins. All the well known properties of Lowicryls were kept and new ones were added
Since the Lowicryl resins were introduced in 1980, numerous papers have reported results obtained by embedding human, animal and plant tissues, or microorganisms such as bacteria or yeast. The protocols used were often optimized to meet specific demands and thus expanded from threat originally proposed by Carlemalm et al. .
To this end, the following protocols may serve as a basis for embedding animal tissues or microorganisms in MonoStep .
Good preservation of cellular and subcellular structures generally requires fixation of the constituting components. Conventionally, fin-structural preservation is achieved by chemical fixation. Often, results obtained with cryofixed material are much better, and at the same time the antigenicity of the embedded specimens is significantly increased. For any biological object the precise conditions for fixation (i.e., the method and, if chemical fixation is employed, the fixative(s) used, its/their concentration and time of fixation) and the sample size have to be optimized and may vary widely among different types of specimens. The effects of different fixatives and different times for fixation on the preservation of antigenicity can be estimated as described by Hortsch et al.  and Roth .
Conventional chemical fixation with aldehydes and osmium tetroxide is performed at temperatures above 0°C [Hayat et al., 1981]. For immunocytochemical purposes, improved fixation was achieved using buffered aldehyde solutions; while Glutaraldehyde primarily preserves cellular fine structure, formaldehyde seems to improve the retention of the antigenicity. In addition, for subsequent dehydration by the progressive lowering of temperature ( PLT ) method, chemical fixation with either formaldehyde or glutaraldehyde or-most frequently - a mixture of both is required. After such chemical fixation and subsequent low-temperature processing (dehydration, resin infiltration, embedding and polymerization), a wide variety of specimens exhibit good structural preservation combined with high immunological reactivity.
With the following standard fixation for cells, we observed good preservation of fine structural details and antigenicity. Fixation is performed for 15-60 min with 0.1-1%(v/v) glutaraldehyde in phosphate-buffered saline (PBS), or with 2% (v/v) formaldehyde/0.1%(v/v) glutaraldehyde in PBS.
For the fixation of mammalian tissue, 10-min perfusion with 3% (v/v) formaldehyde/0.1% (v/v) glutaraldehyde in oxygenated Hank's balanced salt solution is recommended [for details, see e.g. Roth, 1989].
As already mentioned, osmium tetroxide fixation should only be employed if definitely required (e.g., to fix lipid-rich samples), and if so, only at moderate concentrations such as e.d. 0.1% (w/v) for subsequent PLT -dehydration and MonoStep embedding, osmium Tetroxide fixation has to be stopped as soon as the color of the sample turns into a pale yellow since strong UV-absorption of a sample prevents the UV-induced polymerization.
For a general review on fixation, see Bullock .
For immunocytochemical applications, see Leenen et al. , Roth [1982, 1986, 1989], Hobot  (animal tissues), Schwarz and Humbel (microorganisms), or Herman (plant tissues).
The purely physical stabilization of biological structures by rapid-freezing was called cryofixation. It is now well established that cryofixation results in excellent preservation of both, the ultrastructure of the cellular components as well as the antigenicity of biological material. Moreover, cryofixation allows a temporal resolution in the msec range and thus enables to trap and visualize even highly dynamic processes in cells and organelles [Knoll et al, 1987; Heuser et al, 1978]. In contrast, depending on the sample and the fixation protocol used, conventional chemical fixation takes several seconds to minutes to complete.
For many years, most biological samples (particularly tissues) could only be cryofixed in the presence of cryoprotectants such as glycerol or sucrose to decrease the rate of ice crystal formation. However, the current state-of-the-art cryofixation even allows the fixation of completely untreated samples (i.e., no cryoprotectants and/or chemical fixatives are required) at ambient pressure [c.f. Sitte et al., 1987]. The actual quality of cryofixed material [for criteria, see Sitte et al., 1987] is limited by the sample properties (e.g., it size, the heat-transfer rate from the sample to the coolant), the method employed (e.g., slamming or plunging), and the system used. With rapid freezing systems (i.e., plunge, slam, or jet freezer), well-preserved layers of 10-15 µm thickness may be obtained while high-pressure freezing [Moor, 1987; Studer, 1989] may yield up to 600 µm thick layers.
Good results can also reproducibly be obtained with a slam freezer system "Cryoblock" [Escaig,1982] operated at light helium temperature. For cryofixation samples are applied or attached to small pieces of filter or cigarette paper that are then mounted onto slamming supports [Escaig, 1982].
Cryofixed samples can be stored for several months under liquid nitrogen. Prior to low-temperature embedding, they are dehydrated by either freeze-substitioun or freeze-drying [for review, see Plattner and Bachmann, 1982; Robards and Sleytr, 1985; Menco, 1986; Zierold and Steinbrecht, 1987; Müller, 1988]
Biological samples that have been chemically fixed (preferentially using aldehydes) are transferred into a dehydrating agent, generally an organic solvent. To minimize extraction, aggregation, precipitation, or dislocation of cellular components during dehydration, the temperature is decreased stepwise while simultaneously increasing the concentration of the dehydrating agent. It is obviously of crucial importance to prevent the sample from freezing. Therefore, care has to be taken to keep the temperature of the dehydration mixture above its freezing-point. In addition, the dehydrating agent has to be well miscible with the resin used for subsequent embedding. Dehydrating agents that have successfully been used inour laboratory are listed in Table 1.
Concentration Dependence of Solvent Freezing-Points:
Acetone, theanol and methanol are the most commonly used solvents for PLT -dehydration. At each step of the dehydration process, care has to be taken to keep the temperature above the freezing-point of the actual aqueous solvent mixture.
|MonoStep K4M||Polar||Ethanol, methanol, acetone, ethyleneglycol, glycerol, dimethyl formamide|
|MonoStep HM20||Nonpolar||Ethanol, methanol, acetone|
An aldehyde-fixed sample (volume_0.5mm3) in a 10-ml vial is either directly, or after a brief wash in ice cold PBS, transferred into 5ml of the dehydrating agent and processed according to the protocol for PLT -dehydration of biological specimens with ethanol or methanol for subsequent MonoStep embedding.
|For Resin||Concentration of solvent||Time in min||T in°C|
|MonoStep polar||30%||30 min||0|
|MonoStep nonpolar||30%||30 min||0|
During dehydration, the sample should slowly and carefully be agitated from time to time either by hand (gentle swiveling) or using a mechanical stirrer (which should not get in contact with the sample).
Important: The samples should be kept in the same vial during the whole procedure. Any exposure of the sample to air (condensation of ice; drying) has to be avoided (i.e., while changing the dehydrating agent, the sample is kept submerged). The dehydrating agent for the next step of the dehydration protocol has to be added pre-chilled in e.g. 5-ml aliquots. During the dehydration steps, the vials are kept closed by polyethylene lids. After the 100% dehydration step the sample is ready for resin infiltration (see Infiltration).
Dehydration of cryofixed samples in the frozen state by organic solvents at temperatures between -80°C to -90°C is known as freeze-substitution. The frozen water will slowly exchange with the surrounding solvent (e.g, acetone or methanol) that may contain classic chemical fixative(s) (e.g., Glutaraldehyde, osmium Tetroxide, uranyl acetate, or mixtures thereof) [Humbel and Müller, 1986].
The kinetics of the chemical reactions between the fixative(s) and the biological macromolecules are very slow at these conditions [Humbel et al., 1983]. For instance, no significant differences in the preservation of fine structural details were revealed when comparing sections of bacterial cells after freeze-substitution for 85 hours with either pure acetone or 3% Glutaraldehyde in acetone. Freeze-substitution with pure solvents is certainly not a standard procedure and absolutely requires subsequent very low-temperature embedding in LOWICRYL K11M or HM23 resins at temperatures below -60°C [see e.g. Edelmann, 1989; Villiger and Bremer, 1990].
Samples that have been cryofixed as outlined (see Cryofixation) are transferred rapidly from liquid nitrogen to the substitution medium. For routine applications, 0.56 ml Glutaraldehyde of a 70% stock solution in 13 ml of acetone per sample (final Glutaraldehyde concentration: 3% v/v ) can be used. The dehydrating agents should be pre-dried using molecular sieve (0.4nm,). In addition, molecular sieve may also be added to the vial containing the sample during freeze-substitution. The time required for freeze-substitution depends on the physical-chemical properties (e.g., polarity, viscosity) of the organic solvent used (e.g., methanol, ethanol, acetone) and has to be determined experimentally. With microorganisms such as bacteria, 80 to 90 hours at -85°C were sufficient for acetone substitution. After freeze-substitution, the temperature is slowly (5-10°C per hour) raised to the temperature required for resin infiltration (see Infiltration).
For reviews on freeze-substitution, see Robards and Sleytr , Sitte et al. , Humbel and Müller , Steinbrecht and Müller .
In contrast to freeze-substitution, freeze-drying of biological samples followed by subsequent low-temperature embedding has only rarely been used in the past [Wroblewski and Wroblewski, 1986; Chiovetti et al., 1986, 1987; Edelmann, 1986; Steinbrecht and Müller, 1987]. For subsequent X-ray microanalysis and cytochemical analysis, freeze-dried samples may be low-temperature embedding in vacuo in the MonoStep resins polar or nonpolar [Wroblewski and Wroblewski, 1990]
The highly crosslinked polar and nonpolar acrylate-methacrylate-based MonoStep resins are available from Electron Microscopy Sciences (Cat#14335 / 14345 ). MonoStep kits are shipped ready to use.
After dehydration by the PLT method (see section The Progressive Lowering of the Temperature ( PLT ) Method, 3.1. above), biological samples (cultured cells, microorganisms, or small pieces of tissue) are infiltrated by the LOWICRYL resins as outlined below.
|Concentration of the resin||Temperature polar||nonpolar||Time in minutes|
During resin infiltration, the samples are kept in closed 10-ml vials as used for dehydration. Slight agitation from time to time with a toothpick or using a slow rotator is recommended.
Cryofixed samples were infiltrated after freeze-substitution and slow warming (i.e., 5 to 10°C/hour) to the desired temperature according to the following protocol:
|Preparation Step||Temperature Polar||Nonpolar||Time|
|Acetone Wash||-40°C||-50°C||1 hour|
|50% (v/v) resin||-35°C||-50°C||2 hours|
|66% (v/v) resin||-35°C||-50°C||2 hours|
|Pure resin||-35°C||-50°C||2 hours|
|Pure resin||-35°C||-50°C||4 hours|
This protocol requires the presence of chemical fixative(s) during freeze-substitution.
Conventional embedding and polymerization is performed at temperatures ranging between 25°C and 80°C. This requires no special devices. Better preservation of the hydration shell of biological macromolecules [Kellenberger, 1987] may be achieved with low-temperature processing. It is conceivable that this hydration shell plays an important role in determining the antigenicity of biological matter. For low-temperature embedding and polymerization at temperatures ranging from approximately 0°C to -50°C, the use of commercially available freezers is necessary. The sample is rapidly transferred into 0.5-ml gelatin capsules (No. 1, Lilly Col , Indianapolis , IN ; only one sample per capsule!) that are pre-filled with the chilled resin to be used. To minimize contaminations due to compensating moisture, only one capsule should be prepared at a time. To prevent a warming of the resin, both a sample transfer and the filling of the capsule are performed with pre-chilled pipettes (glass or polypropylene). After complete filling and closing of the capsules, they are kept for about one hour in the cold before UV-polymerization is started. Optimal polymerization should be induced by indirect (i.e., reflected) UV-irradiation.
UV Sources from various manufacturers may be used provided their emission maximum is close to 360nm wavelength. To optimize the degree and rate of polymerization of the resin at a fixed temperature, the tube to sample distance may be varied. The polymerization velocity is correct if the resin block after hardening does not show any deformation or bubbles. It has to be kept in mind, that UV-induced polymerization is efficiently inhibited by strongly light absorbing samples (intense color from osmium Tetroxide fixation or dark pigments).
|Resin||Minimal Temperature for Polymerization||Time of UV-irradiation|
After low-temperature polymerization the resin blocks were warmed to room temperature and irradiated for another three days in the direct UV-light, to improve the cutting properties of the blocks.
For ultrathin sectioning of MonoStep-embedded samples, glass or diamond knives with cutting-edge angles of ~35-45°may be used. Ultrathin sectioning of properly polymerized blocks of nonpolar MonoStep resins may be performed as with epoxy-embedded (e.g., EPON 812) samples. However, due to the physical-chemical properties of the acrylates/methacrylates, the contact between the resin and the biological material is much weaker than that with epoxies. Therefore, to avoid pressure-induced deformation in the sample area, the blocks should not be trimmed by hand, but by either a microtome equipped with a glass knife or a special trimming device such as the Ultratrim (Leica Instruments (Reichert-Jung), Vienna , Austria ). The pyramid angle should be about 30°, and the pyramid surfaces should be clean and shiny. The optimal cutting-speed depends on various parameters (e.g., the size and shape of the pyramid, the hardness of both the resin and the embedding biological material, the knife quality) and usually ranges between 2-5mm/s. The resulting sections are deposited on collodion/carbon or Formvar-coated EM grids.
The following precautions are recommended for sectioning specimens that are embedded in the polar MonoStep resins;
Usually, deformed or soft pyramids result from water uptake during sectioning and should therefore immediately be removed from the microtome and dried either in a desiccator or in the presence of a desiccant for at least one day. After that time, the block should be newly trimmed. After sectioning, the polar resin blocks should be stored moisture protected.
The generally low inherent contrast of biological matter upon imaging in the CTEM calls for enhancement by e.g. staining with heavy metal compounds such as uranyl acetate or lead citrate. The procedure for staining conventionally embedded samples [Lewis and Knight, 1974] does also apply for MonoStep sections. However, the physical-chemical properties of the MonoStep resins require different staining times. Using the data given below, good results should be obtained with MonoStep embedded specimens.
|Monostep Resin||Staining Time Uranyl Acetate Lead Acetate or Citrate|
|polar||25-35 min (4-5 min) 1-3 min (45 sec)|
|nonpolar||5-15 min 1-3 min|
EM grids with deposited thin sections are first floated on a drop of uranyl acetate solution for the time indicated, followed by a brief wash, and floated on a drop of lead acetate prepared according to Millonig  or lead citrate. For polar MonoStep sections, the times in brackets have been reported to be sufficient [Roth, 1989; Roth et al., 1990].
Although lead citrate as well as lead acetate can be used as the second staining step, the use of lead acetate [according to Millonig, 1961] gave better results compared to lead citrate staining [according to Reynolds, 1963; Venable and Coggeshall, 1965].
For specimens embedded with the polar MonoStep resin, an uranyl acetate/methyl cellulose staining protocol was developed [Roth, 1989; Roth et al., 1990] and reported to improve the staining of fine structural details in the cytoplasm while being performed much quicker (i.e., ~5 min).
Low-Temperature processing always harbors the danger of unintended temperature fluctuations and the contamination by the condensation of moisture during the various steps involved. Therefore, all instruments should be pre-chilled, and all operations should be performed as quickly as possible! Instruments for sample transfer such as insulated tweezers are commercially available.
Provided good resin infiltration and polymerization are achieved, the different MonoStep resins warrant good cutting properties over the whole low-temperature range.
|Specimen" Milky"||Incomplete dehydration and/or infiltration||• Use smaller samples (0.5mm 3 )
• Prolong infiltration time
• Increase agitation
|Specimen too soft||Incomplete infiltration Sample rich in lipids Incomplete polymerization||• Use smaller samples (0.5mm 3 )
• Prolong infiltration time • Increase agitation
• Change conditions for fixation (include osmium Tetroxide)
• Use smaller samples (0.5mm 3 )
|Resin block too soft||Incomplete polymerization (inhibition by oxygen or water)||• Mix resin components in a stream of nitrogen
• Dry the resin for several hours at room temperature with molecular sieve
• Keep vials or capsules closed
• Close capsules immediately
• Check UV irradiation conditions (source, absorption of sample)
• Repeat polymerization step at room temperature
|Deformed block or bubbles within the block||Polymerization too rapid||• Increase UV source-sample distance
• Reduce light intensity and/or temperature
Immunolabeling is a technique that is performed on thin sections of acrylate based embeddings to localize proteins (in common elements) with respect to the specimen. This can be in common any specific reaction, but in the Immunolabeling it is always an antigen to antibody reaction. A second step involves the visualization of the specific reaction in the electron microscope that involves usually the specific binding of protein A-gold or protein G-gold grains of various size to the F c part of the specific antibody. The dynamic reactivity at pH 7.4 of protein A and protein G with certain species of Immunoglobulins is listed below:
|Immunoglobulin||Protein A||Protein G|
|Mouse IgG1||+ pH9 / - ph 7.4||-|
|Horse IgG (ab)||Weak||+|
|Horse IgG (t)||+||Weak|
In the literature there is a tremendous variety of labeling procedures of which only a selection of standard procedures is listed below.
During a labeling procedure grids with sections are usually incubated for hours on salt solutions that induces oxidation of the copper of standard grids. There are ways to avoid oxidation. The simplest is to protect the copper grids by immersing them in diluted Formvar or collodion before the actual foil is prepared on them. Gold grids can also replace copper. Gold is a soft metal and skill of the operator is needed to handle gold grids. Very widespread is the use of nickel grids. These are ferromagnetic and stick to normal steel forceps. This magnetism also influences the image forming electrons in a microscope in a way that low magnification images are impossible to perform. Also astigmatism correction for high-resolution images on nickel grids is impossible to perform.
Fixation is another topic heavily discussed in the literature. Too strong (even aldehyde) fixation leads to a reduced reactivity of antibody to antigen. Some fixatives like OsO 4 or uranyl acetate cover the antigen preventing a reaction with the antibody (except during freeze-substitution and cryosectioning). These effects can be partially reversed, if needed, by etching (OsO 4 or uranyl acetate) or incubation of the sections (aldehydes) on diluted HCl (pH2) prior to labeling.
Ultrathin methacrylate-(e.g. MonoStep polar and nonpolar) or epoxy (Epon/Araldite, Spurr) sections are transferred with a loop on polylysine coated round cover slips. Residual water is drained with filter paper along the outside of the loop. Note: air-dried resin sections can be stored for months prior to labeling.
After a final wash with buffer, mount coverslips on glass slides using a small drop of mounting medium (Aqua PolyMount or Moviol 4.88) for semi-permanent embedding. The addition of anti-fading agents like DABC) (25-100 mg/ml), Paraphenylenediamine (1 mg/ml) or n-propyl gallate (10 mg/ml) is strongly recommended. Use oil immersion objectives.
Attention: All solutions should be very clean or centrifuged before use for 2 min at 10'000 rpm in an Eppendorf centrifuge.
I would like to thank Dr Heinz Schwarz and Dr. York-Deiter Stierhof from the Ma-Planck-Institute ofr Developmental Biology, Tuebingen ( Germany ) for his contribution of "Immunofluorescence on ultrathin sections" to these application notes.
The LOWICRYL types are (meth-)acrylic acid ester preparations. (Meth-)Acrylic acids can cause allergies.
The following points should always be taken into consideration when working with LOWICRYL products:
The chemical, physical and toxicological properties of these products are not fully known. Avoid contact with skin and eyes. Avoid inhalation of resin vapor. Use well-ventilated fume hood for mixing resins. It has been shown that methacrylate resins can cause irritation to skin and eyes and may cause sensitization to some individuals. The use of disposable utensils and tools is recommended. Note: Kits should not be stored in the refrigerator.
In case of contact, promptly wash affected area of skin with plenty of soap and water. Flush eyes with plenty of water. Get medical attention immediately. After handling, wash thoroughly.
Lowicryl Monostep Embedding Media
Lowicryl Monostep K4M Polar MSDS
Lowicryl HM-20 Monostep Embedding Kit MSDS