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.     

Immunofluorescent Staining of Microtubules in Plant Tissues: Improved Embedding and Sectioning Techniques using Polyethylene Glycol (PEG) and Steedman's Wax

Methods for the indirect immunofluorescent staining of microtubules in embedded and sectioned plant tissues are described and compared. Root tips of Vicia faba, Saccharum officinale, Allium cepa, and root nodules of Glycine max were fixed using conventional methods, embedded in polyethylene glycol or Steedman's wax, sectioned with a glass knife on a rotary microtome, and dewaxed in water or alcohol. The addition of dithiothreitol (DTT), dehydrating at low temperatures and reducing the infiltrations times were found to reduce background fluorescence in Allium cepa. Steedman's wax yields a block that is similar to paraffin and is easier to section than PEG. Routine methods for indirect immunofluorescence were used to stain sections for tubulin/microtubules. The major microtubule arrays of mitotic cells are illustrated in this paper. The principal advantage of this technique is the preservation of cell-to-cell continuity in multicellular tissues. This method provides a much needed technique for the study of the cytoskeleton during growth and differentiation of plant tissues.     

Glycol Methacrylate Embedding of Algmate-Polylysine Microencapsulated Pancreatic Islets

A method for processing and embedding alginate-polylysine microencapsulated pancreatic tissue in glycol methacrylate resin (GMA) is described. Fixation in 4% phosphate buffered formaldehyde, processing in ascending concentrations of glycol methacrylate monomer and embedding in Technovit 7100 results in well preserved morphological details of hydrogels, hydrogel-cell interfaces, and encapsulated pancreatic tissue. Routine staining with Loeffler's methylene blue, hematoxylin and eosin, and Romanovsky-Giemsa gave excellent images of the GMA embedded alginate polylysine membrane and tissues allowing cells on the outside of the capsule to be analyzed effectively as part of the foreign body reaction against the capsule membrane.     

Glycol Methacrylate Embedding of Algmate-Polylysine Microencapsulated Pancreatic Islets

A method for processing and embedding alginate-polylysine microencapsulated pancreatic tissue in glycol methacrylate resin (GMA) is described. Fixation in 4% phosphate buffered formaldehyde, processing in ascending concentrations of glycol methacrylate monomer and embedding in Technovit 7100 results in well preserved morphological details of hydrogels, hydrogel-cell interfaces, and encapsulated pancreatic tissue. Routine staining with Loeffler's methylene blue, hematoxylin and eosin, and Romanovsky-Giemsa gave excellent images of the GMA embedded alginate polylysine membrane and tissues allowing cells on the outside of the capsule to be analyzed effectively as part of the foreign body reaction against the capsule membrane.     

Glycol Methacrylate Embedding in Histotechnology: The Hematoxylin-Eosin Stain as A Method for Assessing the Stability of Glycol Methacrylate Sections

Glycol methacrylate (GMA) samples containing inhibitor in the range of 200-300 ppm were included in a standard embedding mixture. The pH of the GMA samples was measured as a 10% solution of the sample in distilled water. The acidity of GMA due to methacrylic acid causes background staining of sections after basic dyes. The concentration of GMA and the amount of impurities such as methacrylic acid (MA) and ethylene glycol dimethacrylate (EDMA) were measured by gas chromatography. Distinct variations in purity were found among five samples of GMA. Sections derived from GMA samples containing more than 2% EDMA showed few, if any, minifolds after staining with hematoxylin and eosin and were more stable in alcoholic and basic solutions; sections from purer GMA showed minifolds and were less stable. Addition of crosslinkers, EDMA or triethylene glycol dimethacrylate (TEDMA) prevented these artifacts. Crosslinkers clearly influence dimensional changes in sections. Addition of crosslinkers to GMA samples containing minimal amounts of MA improved the results. The possibility of obtaining a high quality GMA embedding medium is discussed.     

Specific Staining of Nucleolar Substance with Aceto-Carmine

A method is described for staining nucleoli intensely by treating tissues with formaldehyde, hydrolysing in normal HC1 at 60°C. and staining with aceto-carmine. With correct hydrolysis time, chromosomes and cytoplasm are almost colorless.

Formaldehyde increases the acidity of cell parts, especially the nucleolus, presumably by neutralizing the basic protein groups, and increases the resistance to hydrolysis, perhaps by protecting the phospholipoprotein complexes which are most abundant in the nucleolus.

Hydrolysis reduces the acidity of cell parts, chiefly by removal of nucleic acids.

Aceto-carmine stains cell structures which are weakly acid in character (about pH 4-5) probably by precipitating as large dye aggregates.

The technic appears to be highly specific for nucleoli and related cell bodies.     

Serial Sectioning of Insects with Hard Exoskeleton by Dissolution of the Exocuticle

The method reported here was designed to produce paraffin serial sections as thin as 5 Mm of insects or other arthropods with a hard cuticle. Heads and abdomens of Apis mellifera, Eristalomyia tenax and Tenebrio molitor were fixed with Schaffer's liquid, dehydrated with 80% ethanol, 90% ethanol, two changes of 100% isopropanol (2 hr each) and 12 hr in a 1:1 mixture of paraffin (58 C melting point) at 60 C. They were molded in paraffin after 12 hr of infiltration under a partial vacuum at 60 C. Large body openings of objects were sealed with paraffin to prevent infiltration of solvents.

Thereafter, the outer paraffin was removed manually and with xylene (15 min); the cuticle was rehydrated with 100% isopropanol and 100% ethanol (15 min each). The objects were then treated with Sputofluol (Merck; a mixture of NaOH and NaCIO) until they became white or their colorless endocuticle was stainable with aniline blue WS (C.I. 42755) after rinsing in a 50% acetic acid solution (v/v). They were then dehydrated with 100% ethanol and 100% isopropanol (15 min each) and subsequently re-embedded in paraffin. They were molded, sectioned, stained and mounted as usual.     

Effects of Storing Initiated 2-Hydroxyethyl Methacrylate Solutions for Embedding Tissues for Light Microscopy: Some Practical Implications

The effects of storing 2-hydroxy-ethyl methacrylate (HEMA) solutions for embedding tissues for light microscopy were studied using three commercially available HEMA embedding kits: Technovit 7100, Technovit 8100, and JB-4. These HEMA solutions were examined at various times of storage over a period of one year using a panel of physicochemical techniques including gas chromatography, tltration, viscosimetry, determination of the maximum polymerization temperature and the time required to reach the maximum temperature, and detection of degradation products of HEMA monomers by histochemical procedures. The quality of the resin blocks was examined by the observation of mini-folds in sections. Data obtained from these tests showed that the release of by-products as a result of the degradation of the HEMA monomer during storage of HEMA solutions does not occur. Development of cross-linking agents by transesterification of HEMA monomer was not detected either. Gradual decrease of the inhibitor concentration during storage proved to be the main cause of the reduction of shelf-life of HEMA solutions. Inconsistent tissue infiltration after storage may be due to decreased rates of tissue penetration as a result of HEMA chain lengthening. Guidelines for safe and economical handling of HEMA mixtures are given.     

植物材料快速石蜡制片方法

真空干燥箱已越来越广泛地应用于现代生物学研究领域。该文利用真空干燥箱温度和负压的可控制性能,将固定、脱水、透明和石蜡渗透等过程在真空干燥箱中进行,建立起一套可行的植物组织快速石蜡制片方法。结果显示,真空干燥箱的应用加速了多种试剂的渗透速率,提高了切片质量,达到了优化实验步骤、节省实验时间和减少室内有毒化学气体污染的目的。     

Computing Projections into Cones Generated by a Matrix

A method is described for computing orthogonal projections into finitely generated cones. This method can be used to solve nonnegative least squares approximation problems, to find the multivariate onesided Maximum Likelihood estimator and also determines the most stringent somewhere most powerful test of Schaafsma. The gist of the procedure is the unconstrained maximization of a numerically simple function. This function has a global maximum and allows an uncomplicated maximum search since local maxima do not exist. The maximum can be obtained after a finite number of iterations.     

Embedding of Neural Tissue in Agarose or Glyoxyl Agarose for Vibratome Sectioning

Agarose was used to embed the brain or spinal cord of lampreys or rats before cutting vibratome sections. Agarose embedding was compatible with immunocytochemistry or the use of horseradish peroxidase as a neuroanatomical tracer. Concentrated agarose with high intrinsic gel strength was optimal for embedding glutaraldehyde fixed neural tissue. A quick procedure was to blot tissue and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in 50 mM neutral-pH TFUS buffer before cutting 50-100 μm vibratome sections. An alternative procedure that improved retention of tissue sections in the agarose was to rinse the tissue in H₂0, blot and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in H₂0, then equilibrate the block overnight in buffer. Phosphate buffer prevented complete dissolving of agarose. Tissue could be covalently linked to the embedding matrix using a novel aldehyde-derived agarose (NuFix® FMC BioProducts). Slices of spinal cord from neonatal rats could be cut after embedding in 5% FMC Seaprep® agarose in rat Ringer's at 23-26 C.     

Improvement in the Histochemical Demonstration of Acid Phosphatase, β-Galactosidase and Nonspecific Esterase in Glycol Methacrylate Tissue Sections by Cold Temperature Embedding

A simple, cold-embedding method (on cracked ice at 2 C) is presented for the demonstration of acid phosphatase, β-galactosidase and nonspecific esterase in glycol methacrylate tissue sections.     

Neutralization of Acid in Glycol Methacrylate and the Use of Cyclohexanol as a Plasticizer

A method for neutralizing acidic compounds present in commercial samples of glycol methacrylate is described. Dimethylaminoethylmethacrylate is added to the glycol methacrylate, the amount required being determined by titration. Further improvement in staining of tissues embedded in the neutralized methacrylate can be brought about by the use of cyclohexanol as a plasticizer.     

Immunohistochemical Detection of Epidermal Growth Factor in Glycol Methacrylate Embedded Tissue

This study addresses a variety of immunohistochemical conditions for detecting EGF in 3.5% paraformaldehyde fixed, glycol methacrylate embedded tissue including antigen unmasking with trypsin, dilution of primary antibody, and incubation time with primary antibody. Color development was achieved with a biotinylated secondary antibody linked to an avidin biotinylated peroxidase complex. Trypsinization and a 12 hr incubation with the primary antibody was essential to detect EGF in this system. Adequate staining could be achieved with a 1:100 dilntion of the primary antibody.     

Application of Stains-All for Demarcation of Cement Lines in Methacrylate Embedded Bone

Cement lines provide a record of sites of past remodeling buried in the matrix of bone. A method is reported for application of Stains-all, a cationic carbocyanine dye, for demarcation of cement lines in bone. The method, which is simple, works well for both glycol methacrylate and methyl methacrylate undemineralized embedments and produces good concomitant staining of cytoplasm and nuclei of osteoblasts, osteoclasts and marrow cells.     

Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand

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Secreted phospholipase A2 type II is present in Paget's disease of bone and modulates osteoclastogenesis,apoptosis and bone resorption of human osteoclasts independently of its catalytic activity in vitro

ObjectivesTo study the role of secreted phospholipase A₂ (sPLA₂) in the pathophysiology of human osteoclasts (OCs).MethodsImmunohistochemistry and sPLA₂ inhibitors were to determine the localization of sPLA₂ and its role in OCs biology.ResultssPLA₂ is expressed by OCs from healthy fetal bone and OCs from Paget's disease but not in normal bone. Inhibition of sPLA₂ greatly reduces in vitro osteoclastogenesis.DiscussionThe decrease in OCs formed could be attributed to a decline in the viability of CD14⁺ OC precursors as well as a reduced viability of mature OCs. Inhibition of sPLA₂ strongly decreases bone resorption by OCs independently of actin cytoskeleton remodeling, probably also by reducing OCs viability.ConclusionHigh amounts of this enzyme are present in fetal and Pagetic bone samples. Inhibition of sPLA₂ in vitro decreases osteoclastogenesis and OC activity and might constitute an interesting pharmacologic target for diseases with high bone turnover.     

Guided bone regeneration produced by new mineralized and reticulated collagen membranes in critical-sized rat calvarial defects

The aim of this study was to evaluate the bone regenerative effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen membranes in critical-sized defects on rat calvarias. Bone calvarial defects were induced in Wistar rats, which were then divided into five groups: a sham group; a control group, which received a commercial membrane; and GA, 25GA, and 75GA groups, which received one of three different polyanionic collagen membranes mineralized by 0, 25, or 75 hydroxyapatite cycles and then cross-linked by GA. Bone formation was evaluated based on digital radiography and computerized tomography. Histological analyses were performed 4 and 12 weeks after the surgical procedure to observe bone formation, membrane resorption, and fibrous tissue surrounding the membranes. Measurement of myeloperoxidase activity, tumor necrosis factor alpha, and interleukin 1beta production was performed 24 h after surgery. The percentage of new bone formation in the GA, 25GA, and 75GA groups was higher compared with the control and sham groups. In the GA and 25 GA groups, the membranes were still in place and were contained in a thick fibrous capsule after 12 weeks. No significant difference was found among the groups regarding myeloperoxidase activity and interleukin 1beta levels, although the GA, 25GA, and 75GA groups presented decreased levels of tumor necrosis factor alpha compared with the control group. These new GA cross-linked membranes accelerated bone healing of the calvarium defects and did not induce inflammation. In addition, unlike the control membrane, the experimental membranes were not absorbed during the analyzed period, so they may offer advantages in large bone defects where prolonged membrane barrier functions are desirable.