Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Improved biocytin labeling and neuronal 3D reconstruction

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.     

A Celloidin Infiltration-Frozen Section Sequence for Enhanced Preservation of Phosphatases in Bone

The effect of the following embedding procedures on the acid and alkaline phosphatase content of decalcified mouse tibiae has been studied: embedding in 23% gelatine for 18 hr at 37° C, embedding in paraffin wax in vacuo for 1 hr at 58° C, and impregnation with 4% celloidin in diethyl ether and ethanol at 4° C for 2-3 days. Unsupported tissues were also used to demonstrate these enzymes for comparison with the above procedures. Tibiae were first fixed in 10% neutral formalin at 4° C for 15 hr, decalcified in equal volumes of 2% formic acid and 20% sodium citrate at pH 4.9 for not more than 5 days and then washed in distilled water before carrying out the embedding schedules. The celloidin-impregnated tibiae were placed in 70% ethanol to harden the celloidin and then washed in distilled water for 1-2 hr. These tibiae and those embedded in gelatine were cast in a gelatine block which was then hardened in 10% neutral formalin at 4° C for 2 hr. Sections of these and unsupported tibiae were cut at 15 μ on a freezing microtome. Decalcified tibiae embedded and blocked in paraffin wax were sectioned at 15 μ on a base sledge microtome. The enzymes were demonstrated using the coupling azo dye method given by Pearse (Histochemistry, 1st Ed. 1954). The stable diazotates of 4 benzoyl amino 2-5 diethoxyanilene, 3 nitro toluidine and o-dianisidine were used. Of the embedding procedures paraffin wax embedding produced the greatest loss of both enzymes. Gelatine embedding and infiltration with celloidin were equally good for the demonstration of acid phosphatase but for alkaline phosphatase the celloidin method was superior. The gelatine embedded material did not produce consistently good results. Celloidin-impregnated tibiae could be stored without marked deterioration of the enzyme content for longer than gelatine-embedded tibiae.     

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.     

An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

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.     

An embedding method for histochemical studies of undecalcified skeletal growth plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tisssues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

Piccolyte investments for better section cutting.

Piccolyte 115 (beta-pinence polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

A methacrylate embedding procedure developed for immunolocalization on plant tissues is also compatible with in situ hybridization.

A recently described method that uses methacrylate embedding of aldehyde fixed plant tissues allows the immunolabelling of a range of antigens (Baskin et al. 1992). We have tested whether the same embedding procedure is also compatible with in situ hybridization. For this purpose we have used 2- 5 μm sections of methacrylate embedded plantlets of Arabidopsis thaliana. After removal of the resin the sections were prepared for in situ hybridization following standard procedures. Three different digoxygenin (dig)-labelled probes were used, recognizing RNAs coding for the chlorophyll a/b binding protein cab-140, the β-tubulin tub5 and meri a member of the meri-5 family. Each of the probes shows the labelling pattern expected from the literature. Moreover, the method allows a good structural preservation of very fragile tissues, in contrast to paraffin embedding. We conclude that methacrylate embedding, allowing both immunolabelling and in situ hybridization with high resolution and structural preservation, offers a high potential for the functional analysis of genes and proteins in plant development. This is especially true for Arabidopsis thaliana, a widely used model species where it seems to be the method of choice.     

Piccolyte Investments for Better Section Cutting

Piccolyte 115 (β-pinene polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of paraffin infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

New aspects of bacterial ultrastructure as revealed by modern acrylics for electron microscopy

Modern acrylics can be used over a wide temperature range (+60 degrees C to -80 degrees C) for infiltration, embedding, and polymerization. They can be used in procedures involving chemical fixation or rapid freezing. This flexibility allows for studies to be carried out upon the effects that different parameters involved in preparing biological tissue for microscopy have upon structure and retention of immunoreactivity. With most preparative methods contributions have been made to our knowledge on bacterial structure in gram-negative and gram-positive cells. The future should lie in integrating the advantages of the various methods for the purpose of advancing our understanding of bacterial structure/function.     

Rapid fixation and embedding for electron microscopy

A method has been developed for rapid processing of animal tissues for electron microscopy. The whole process of fixation staining dehydration, infiltration and embedding including polymerization is completed in less than 4 hr. A variety of human and animal tissues such as liver, spleen, muscle, kidney and embryonic chick heart were processed by this method and the results were excellent. The rapid fixation and embedding method is strongly recommended when relatively soft tissues are to be studied. This method is especially useful for examining pathological tissues for rapid diagnostic purposes.     

Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life

Background

Lens crystallines are special proteins in the eye lens. Because the epithelial basement membrane (lens capsule) completely encloses the lens, desquamation of aging cells is impossible, and due to the complete absence of blood vessels or transport of metabolites in this area, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its ¹⁴C content from the atmospheric carbon dioxide. The ¹⁴C content of the lens proteins thus reflects the atmospheric content of ¹⁴C when the lens crystallines were formed. Precise radiocarbon dating is made possible by comparing the ¹⁴C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric ¹⁴C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on a yearly basis this allows very accurate dating.

Methodology/Principal Findings

Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The close relationship may be further expressed as a mathematical model, which takes into account the timing of the crystalline formation.

Conclusions/Significance

Such a life-long permanence of human tissue has hitherto only been described for dental enamel. In confront to dental enamel it must be held in mind that the eye lens is a soft structure, subjected to almost continuous deformation, due to lens accommodation, yet its most important constituent, the lens crystalline, is never subject to turnover or remodelling once formed. The determination of the ¹⁴C content of various tissues may be used to assess turnover rates and degree of substitution (for example for brain cell DNA). Potential targets may be nervous tissues in terms of senile or pre-senile degradation, as well as other highly specialised structures of the eyes. The precision with which the year of birth may be calculated points to forensic uses of this technique.     

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     

A Tissue Basket for Paraffin Infiltration

The processes of washing, dehydration and paraffin infiltration when large numbers of tissues must be individually identified are always laborious. Washing and dehydration can be carried out in individual glass vials with relative ease and fairly rapidly if gravity flow reagent bottles are used to fill the vials. The use of similar vials for paraffin infiltration is usually complicated by hardening of the paraffin if too many vials are removed from the oven at once, or by cooling of the oven itself if it is too frequently opened. The same criticism applies to the process of embedding tissues when large numbers of vials must be handled simultaneously.     

A Tissue Basket for Paraffin Infiltration

The processes of washing, dehydration and paraffin infiltration when large numbers of tissues must be individually identified are always laborious. Washing and dehydration can be carried out in individual glass vials with relative ease and fairly rapidly if gravity flow reagent bottles are used to fill the vials. The use of similar vials for paraffin infiltration is usually complicated by hardening of the paraffin if too many vials are removed from the oven at once, or by cooling of the oven itself if it is too frequently opened. The same criticism applies to the process of embedding tissues when large numbers of vials must be handled simultaneously.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

Transport processes of fluid exchange in the crystalline lens of the rabbit eye

Processes of fluid exchange in the crystalline lens of the rabbit eye were investigated. The direction of movement of fluid in the crystalline lens was investigated from the movement of fluorescein by the method of "stopped diffusion". It has been found that the mechanism of fluid transport in the crystalline lens is active and is carried out by means of the Na-kappa-ATPase transport system. The energy necessary for the active transport of fluid inside the crystalline lens is in the range (1.5-6) x 10(-2) J. Owing to the active fluid transport, the pressure inside the crystalline lens constantly increases by 6 mm Hg. In rabbit's life-time, the movement of fluid in the crystalline lens occurs in the direction from the anterior to the posterior surface followed by the exit to vitreous humor.     

Better epoxy resin embedding for electron microscopy at low relative humidity.

In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used; impregnate tissues using the following solutions 1) 70% alcohol - 5 minutes, 2) 95% alcohol - 2 x 15 minutes, 3) absolute alcohol - 3 x 20 minutes, 4) acetone - 2 x 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 812, 39.4 g; DMP-30, 2.6 g) - 1 hour, 6) acetone-epoxy resin 1:3 - 1 hour, 7) epoxy resin - 1 hour; complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.