Serial sectioning techniques for a modified LKB Historesin

A glycol methacrylate-based plastic that is capable of producing serial sections has been introduced by LKB. This plastic, provided in the LKB 2218-500 Historesin Embedding Kit, has been tested in our laboratory for its ribbon forming capacity. Various block sizes, concentrations of the softening agent polyethylene glycol 400 (PEG), and tissue types have been examined to determine the optimal conditions for ribbon formation. Although unmodified LKB Historesin is capable of forming ribbons, these ribbons often break. The addition of PEG to the embedding solution enhances ribbon formation. When sectioning with glass knives the best results are achieved with the addition of 0.2 ml of PEG/5.0 ml of embedding medium. A conventional AO rotary microtome can be used to produce ribbons if, in addition to the added PEG (optimal concentration 0.25-0.30 per 5 ml of embedding medium) a thin layer of dental wax is added to the upper and lower surfaces of the block. Ribbons form more easily on microtomes, such as the LKB Historange, that have a retractable specimen arm. If serial sections are to be produced it is very important that the upper and lower faces of blocks be parallel.     

Serial Sectioning of Tissues in Glycol Methacrylate by Supplementary Embedding in a Paraffin-Plastic Matrix

Glycol methacrylate, while offering certain advantages over paraffin as an embedding medium, is difficult to use because it will not ribbon. Rohde (1965) developed a method for producing ribbons of methacrylate sections, but we had little success with it because the ribbons tended to fall apart when even slight stresses were applied to them. We have therefore made use of the principle of double embedding, as this has been used for obtaining serial sections of material embedded in nitrocellulose.     

Serial Sectioning of Tissues in Glycol Methacrylate by Supplementary Embedding in a Paraffin-Plastic Matrix

Glycol methacrylate, while offering certain advantages over paraffin as an embedding medium, is difficult to use because it will not ribbon. Rohde (1965) developed a method for producing ribbons of methacrylate sections, but we had little success with it because the ribbons tended to fall apart when even slight stresses were applied to them. We have therefore made use of the principle of double embedding, as this has been used for obtaining serial sections of material embedded in nitrocellulose.     

A Method for Making Ribbons of Semithin Plastic Sections

Epoxy resin sections form strong, heat resistant ribbons if, prior to sectioning, contact cement has been painted onto the leading and trailing faces of the block. The forming ribbon floats onto a drop of water held in place by a wax line drawn across the back of the glass knife parallel to the cutting edge. A long trough made from stainless steel tubing is inserted horizontally into the drop, and as the ribbon lengthens it is directed into the trough. The ribbon can be carried in the trough to a hot plate for expansion and then poured onto a slide for mounting. The serial ribbons obtainable by this simple procedure greatly facilitate three dimensional reconstruction of fine tissue structures.'     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

N-Butyl methacrylate and paraffin as an embedding medium for light microscopy.

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18--24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.     

A Plastic Embedding Medium for Thin Sectioning in Light Microscopy

Tissue blocks with surface areas up to 2 cm² can be sectioned at 1 or 2 μ after embedding in a medium consisting of: methyl methacrylate, 27 ml; polyethylene glycol distearate MW 1540, 6 gm; dibutyl phthalate, 4 ml; and Plexiglas molding powder A-100, 9 gm (added last). The methacrylate mixture is polymerized at 50° C by benzoyl peroxide, 0.8 gm/ 100 ml of methacrylate. The polymerized matrix is transparent and the blocks can be cut on a rotary microtome with a steel knife. The plastic can be removed from sections with acetone prior to staining. Artifacts caused by embedding and sectioning are negligible     

Effect of Additives on the Production, Viability and Virulence of Blastospores of Metarhizium anisopliae

Studies on the blastospore production of Metarhizium anisopliae var. anisopliae were conducted in Adamek's medium used as a standard, enriched with lecithin, collagen, lactic acid or polyethylene glycol 200 (PEG 200) to increase spore yield and suppress mycelial pellet formation. The addition of 5% lecithin resulted in a significant 10-fold increase in spore yield up to 1.9 108 blastospores/ml compared with 1.9 107 spores/ml in the standard medium. Collagen (3%) increased the number of blastospores 3.7-fold, and lactic acid (1.5%) two-fold. A reduction of mycelial pellet formation in favour of spore production was noted with each additive. The viability of blastospores at 40IC from media with lecithin, collagen and lactic acid suspended in 25% Ringer's solution was comparable to that of spores produced in the standard medium. Striking differences were noticed in the viability of spores produced with 5% PEG 200 in standard medium. The half-life of blastospores produced in standard medium suspended in sunflower oil was 33.6 h and that of 5% PEG 200 spores only 25.2 h. In bioassays, the virulence of spores produced in standard medium to which 3% lecithin, 3% collagen, 1.5% lactic acid or 5% PEG 200 had been added was tested against third-instar nymphs of Locusta migratoria migratorioides (R. & F.). The median lethal time and the mortality of L. migratoria achieved with blastospores produced with 3% lecithin (5.7 days, 99%) was comparable to that of blastospores from standard medium (5.1 days, 98%). The virulence of blastospores from all other media with additives was significantly reduced.     

Improved Polyester Wax Embedding for Histology

Polyester waxes are fatty add esters of polyethylene glycol. Polyethylene glycol 400 distearate melts at 35°C, infiltrates tissues well, and sections readily at 2 μ to more than 30 μ. Sections 2 μ to 6 μ are more easily cut when a kitchen strainer full of solid CO₂ (dry ice) is mounted above the microtome to cool the block and the knife, and when the knife crosses the block very slowly. Ribbons are flattened in water at room temperature and are mounted conventionally. Polyester ribbons are somewhat stickier than paraffin ribbons. Polyethylene glycol 400 distearate is slightly hydrophilic; immediately after microtomy and before the ribbon is affixed to the microscope slide, sections in the wax ribbon may conveniently be stained with 0.05% toluidine blue in aqueous benzoate buffer, pH 4.4. Tissue structure is better preserved in polyester than in paraffin wax, probably because structural lipids are better retained and localized. However, this difference between waxes is slight if tissues are well fixed and dehydrated. Other advantages of polyester wax are that sections fragment less, hard tissues rarely split away from the wax ribbon, no static electricity is generated, and the microtome knife seems to remain sharp for a longer time.     

Serial Sections of Cooked Rice Embedded in Carbowax

Serial sections of cooked rice kernels may be obtained by following either of two dehydration schedules and embedding in Carbowax. In the first schedule the cooked, rinsed and drained kernels are immersed several days in a nonaqueous fixative composed of: isopropyl alcohol, 10 ml; propionic acid, 30 ml; acetone, 10 ml; methylal, 40 ml; dioxane, 30 ml; and propylene glycol, 30 ml (Newcomer's, modified), followed by 7 or 8 days in equal parts of propylene glycol, dioxane and glycerol (changed once), and 4 days on a warming table in the same mixture with 5% Carbowax added. The dehydrated kernels are then infiltrated 4-24 hr with a Carbowax embedding mixture. In the second schedule they are immersed several days in an aqueous solution consisting of: propylene glycol, 12.5 ml; polyethylene glycol 400, 12.5 ml; either with 75 ml of water containing 0.1% thymol, or with a mixture of water, 65 ml; formalin, 10 ml; CaCl₂, 1 gm; and CdCl₂, 1 gm; followed by 3 or 4 days in 50% propylene glycol, and 3 or 4 days on a warming table in 80% propylene glycol with 5% Carbowax added. Infiltration is as above. The composition of the embedding mixture is varied according to the temperature and humidity likely to prevail during sectioning. The texture of the wax may be improved by adding small amounts of gum arabic, spermaceti, and glycerol. Serial sections 3-10 μ thick are placed on clean dry slides, and adhesive dropped at the edges of the ribbon of Carbowax until it is dissolved. The adhesive consists of water-glass (concentrated solution), 1 ml; concentrated ammonia, 1 ml; Carbowax, 5 gm; and water, 98 ml. After the slides are dry they are stored, or immersed 10 min in chloroform, collodionized, and passed to staining solutions. Atmospheric conditions affect not only the Carbowax, but also the response to reagents of cooked rice and of sections.     

A possible role for the dimer ribbon state of scallop sarcoplasmic reticulum. Dimmer ribbons are associated with stabilization of the Ca(2+)-free Ca-ATPase

The Ca-ATPase activity of membranous scallop sarcoplasmic reticulum was found to be unstable when the Ca(2+)-binding sites on the Ca-ATPase were unoccupied. The decay in activity could be slowed or halted by inclusion in the preincubation medium of Na+, K+, nucleotides, ethylene glycol, or high concentrations of choline chloride. Stabilization of the Ca(2+)-free Ca-ATPase by Na+ and K+ showed a markedly different concentration dependence to that seen with activation of the Ca(2+)-activated ATPase activity by the two ions. Examination in the electron microscope of scallop membranes negatively stained in the presence of EGTA under conditions where the enzyme had been stabilized against lack of Ca2+ always showed vesicles containing dimer ribbon structures, whereas unstabilized membranes did not show dimer ribbons. There was an association between the effectiveness of a medium in stabilizing the enzyme in the presence of EGTA and the extent and quality of the dimer arrays seen in the microscope. Comparison of the range of Ca2+ concentration over which the Ca(2+)-binding sites on the scallop Ca-ATPase titrated with the range over which the dimer ribbon structural state was lost indicated that the Ca(2+)-binding sites on the Ca-ATPase must be empty for dimer ribbon formation to occur. Previous studies (Franzini-Armstrong, C., Ferguson, D. G., Castellani, L., and Kenney, L. J. (1987) Ann. N. Y. Acad. Sci. 483, 44-56) have found that the Ca-ATPase molecules in scallop adductor muscle freeze-fractured after fixation under relaxing conditions are arranged in dimer ribbons. Thus, the association of stabilization of the Ca(2+)-free Ca-ATPase with the presence of dimer ribbons implies that one function of the dimer state may be to stabilize the scallop enzyme in situ, when the Ca2+ concentration in the sarcoplasm is low and the muscle is relaxed.     

Floating-Out Technics for Rapid Placement of Ribbons of Serial Sections on Slides

The floating-out technic, popular for single paraffin sections, can be applied successfully to ribbons of serials by either of two procedures. (1) If spreading time for the sections is uncritical suitable lengths of ribbon for attachment to a slide are laid on water at a temperature about 8° C below the melting point of the paraffin and manipulated with a rubber bulb pipette to form a unit. This ensemble can then be picked up by the slide in much the same manner as a single section. (2) If spreading time is critical, as for objects that have had guide limes embedded with them, several ribbons are arranged on a cold, dry slide and transferred to the water as a unit. Placing the ribbons on the cold slide so that they slightly overhang one end and the sides of the slide allows them to make proper contact with the water as the slide is immersed. To facilitate controllable spreading in both methods, the water should have added to it 0.5 ml of albumen-glycerol adhesive per 100 ml. Adding water to the slide after the sections have been picked up or manipulation of the ribbons is generally unnecessary if the ribbons have been aligned accurately on the floating-out bath.     

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.     

Viability,cell division and microcallus formation of barley microspores in culture

Barley microspores were viable when cultured in a sugarless medium. Adding 2g of glucose to 1l of this medium resulted in a significant reduction in the frequency of viable microspores. The frequency of viable microspores was further reduced when 50g of cellobiose, glucose, maltose, melezitose, raffinose or sucrose were added to 1l of the culture medium containing 2g/l glucose. Adding 50g of melibiose, Ficoll, polyethylene glycol (PEG) or a combination 50g each of Ficoll and PEG to 1I of the medium containing 2g/l glucose had very little effect on the viability of the microspores.Up to 66% of the viable microspores were able to divide and many of these developed into microcalli in the basal medium complemented with melibiose, maltose, melezitose, raffinose, Ficoll, PEG or a combination of Ficoll with PEG. Sucrose, cellobiose and glucose added in large quantities inhibited cell division in microspores or destabilized the microspores and only very few of them developed into microcalli.The microcalli in the PEG, Ficoll, Ficoll-PEG and melibiose media were smaller in size than those grown in the melezitose, maltose and raffinose media. Sustained cell division and microcallus formation were observed in a medium with melibiose or maltose as sole source of sugars.Abbreviations 2.4-D 2,4-dichlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - PEG Polyethylene glycol     

Synaptic arrays of the inner plexiform layer in the developing retina of Xenopus

During embryonic and larval development in Xenopus laevis, arrays of synapses made by amacrine cells form in two phases: an initial phase of rapid synaptic addition and a second phase of slower addition. In the region near the optic nerve, at which all measurements were made, these synapses first appear at stage 40 (approx 66 hr postfertilization). Connectivity increases at a rate of 8.6 synapses per hr per inner nuclear layer (INL) nucleus until stage 47 (132 hr postfertilization). After this phase the rate of formation decreases to 1.19 synapses per hr per INL nucleus. Synaptic arrays made by bipolar cells have only one phase of addition. A synapse made by a bipolar cell may be identified by its presynaptic ribbon, the first of which are seen at stage 40. Ribbons are added to the IPL neuropil at a rate of 4.6 ribbons per hr per INL nucleus until stage 47. After this the number of ribbons per INL nucleus in the area near the optic nerve remains constant. Although they may be found, amacrine to amacrine synapses (serial conventional) remain at low numbers throughout larval and early postmetamorphic life. This is unlike the condition found in Rana pipiens where a dramatic increase in amacrine to amacrine connectivity occurs at metamorphosis.     

Polyethylene glycol embedded tissue sections for immunoelectronmicroscopy

Summary Several methods for tissue embedding in polyethylene glycol (PEG) were compared with regard to their applicability for pre-embedding immunoelectronmicroscopy. Existing embedding procedures gave unsatisfactory results and therefore a modified procedure was developed. This method, consisting of very brief tissue infiltration with PEG 1500, to which 3% water is added, allowed adequate tissue sectioning. Using these sections for preembedding immunoelectronmicroscopical localisation of glucagon in bovine pancreatic islets adequate ultrastructural morphology was obtained in combination with excellent preservation of peptide hormone immunoreactivity.Supported in part by grant no. PAL 52-77 of the Queen Qilhelmina Cancer Foundation     

Hydroxyethyl Methacrylate Combined with Polyethylene Glycol 400 and Water; an Embedding Medium for Routine 1-2 Micron Sectioning

Pieces of tissue, with the largest dimension not exceeding 7 mm, are fixed and dehydrated by the procedures of choice. Two stock solutions: A, for infiltration; and B, the accelerator, are used in embedding. Formulas: A, 80 ml of glycol methacrylate (2-hydroxyethyl methacrylate—Rohm and Haas Co., Philadelphia, Pa.) is mixed well with 12 ml of polyethylene glycol (Carbowax) 400 and 8 ml of water; then 0.27 gm of benzoyl peroxide added, heated to dissolve the peroxide, and allowed to cool to room temperature. B, polyethylene 200 or 400, 15 parts, and N,N-dimethylaniline, 1 part, mixed thoroughly. Tissues are first infiltrated completely with solution A, then cast in a mixture consisting of 42 parts of A mixed with 1 part of B. Polymerization occurs in 45 min to 3 hr, depending on the temperature. In a water bath at 20 C, the time required was found to be about 3 hr; at 25 C, 1.5 hr; and at 30 C, 45 min. The plastic block can be trimmed easily, and sections 1-2 μ thick readily cut. Sections can be attached to slides by water flotation, without adhesive, and should be dried at room temperature. Staining with aqueous solutions of basic and acid dyes, without removing the embedding matrix, is sharp and brilliant. When staining of the matrix by basic dyes occurs, this background stain can be completely removed by differentiating in either 2-butoxyethanol, pure ethanol, or a mixture of the two. A number of histochemical reagents have been found compatible with this embedding procedure.     

Ribbon Synapse Plasticity in the Cochleae of Guinea Pigs after Noise-Induced Silent Damage

Noise exposure at low levels or low doses can damage hair cell afferent ribbon synapses without causing permanent threshold shifts. In contrast to reports in the mouse cochleae, initial damage to ribbon synapses in the cochleae of guinea pigs is largely repairable. In the present study, we further investigated the repair process in ribbon synapses in guinea pigs after similar noise exposure. In the control samples, a small portion of afferent synapses lacked synaptic ribbons, suggesting the co-existence of conventional no-ribbon and ribbon synapses. The loss and recovery of hair cell ribbons and post-synaptic densities (PSDs) occurred in parallel, but the recovery was not complete, resulting in a permanent loss of less than 10% synapses. During the repair process, ribbons were temporally separated from the PSDs. A plastic interaction between ribbons and postsynaptic terminals may be involved in the reestablishment of synaptic contact between ribbons and PSDs, as shown by location changes in both structures. Synapse repair was associated with a breakdown in temporal processing, as reflected by poorer responses in the compound action potential (CAP) of auditory nerves to time-stress signals. Thus, deterioration in temporal processing originated from the cochlea. This deterioration developed with the recovery in hearing threshold and ribbon synapse counts, suggesting that the repaired synapses had deficits in temporal processing.