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.     

The lipid solubility of fixative,staining and embedding media,and the introduction of LX-112 and Poly/bed-812 as dehydrants for epoxy resin embedment

The manufacture of Shell, Epon-812 (E-812) resin has recently been discontinued. E-812 and two newly introduced E-812 substitutes, the Ladd-112 (LX-112) and the Polysciences, Poly/bed-812 (PB-812) resins, were studied biochemically and morphologically for their effectiveness as polar dehydrants. Their technical properties as general E-812 replacements were also explored. In the biochemical studies, acetone was more effective in retaining lung phospholipid components than ethanol, and resin dehydration was more effective than either acetone or ethanol. There was no appreciable difference in lipid solubility among the three resins. Acetone and uranyl magnesium acetate each had a loosening effect on previously fixed phospholipids. The PB-812 and E-812 resin dehydrated blocks of dense animal tissues, demonstrated serious technical difficulties during sectioning. The L-112 resin substitute, due to its low viscosity and improved infiltration, was found to be technically as effective a dehydrant as ethanol or acetone. None of the three resins was successful as dehydrating agents for the plant tissue. With organic solvent dehydration, both epoxy resin substitutes demonstrated excellent embedment properties with both animal and plant tissues.     

The influence of embedding media and fixation on the postembedment ultrastructural demonstration of complex carbohydrates. III. High iron diamine staining for sulfated glycoconjugates

The high iron diamine (HID) method for detection of sulfated complex carbohydrate has been applied directly on thin sections of variably fixed tissues embedded in epoxy and nonepoxy resins. Results with postembedment HID staining in mouse intestinal epithelium are compared to those previously obtained using preembedment methods. Sections from epoxy-embedded tissues have been found to exhibit the weakest staining intensity. Intense, specific staining was obtained in tissues not postfixed with osmium tetroxide and embedded in polystyrene, polyester resins, styrene-methacrylate, and especially the styrene-Vestopal W embedding mixture. Postosmication of tissues abolished HID staining in epoxy resins and the styrene-Spurr's resin embedding mixture, but only reduced the staining intensity in tissues embedded in nonepoxy resins.     

Epoxy Resins in Electron Microscopy

A method of embedding biological specimens in araldite 502 (Ciba) has been developed for materials available in the United States. Araldite-embedded tissues are suitable for electron microscopy, but the cutting qualities of the resin necessitates more than routine attention during microtomy. The rather high viscosity of araldite 502 also seems to be an unnecessary handicap. The less viscous epoxy epon 812 (Shell) produces specimens with improved cutting qualities, and has several features—low shrinkage and absence of specimen damage during cure, minimal compression of sections, relative absence of electron beam-induced section damage, etc.—which recommends it as a routine embedding material. The hardness of the cured resin can be easily adjusted by several methods to suit the materials embedded in it. Several problems and advantages of working with sections of epoxy resins are also discussed.     

Immunoelectron microscopic labeling of immunoglobulin in plasma cells after osmium fixation and epoxy embedding

Previous studies have found that immunoglobulin cannot be immunolabeled in tissues prepared for electron microscopy by usual methods. To test this conclusion, we used a protein A-gold postembedding immunolabeling method on tissues that were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epoxy resin; sections were pretreated with sodium metaperiodate. A variety of common fixation protocols were also used and the most suitable conditions for immunolabeling were determined. This technique permitted the ultrastructural localization of immunoglobulin light chains in optimally preserved and contrasted plasma cells from human tonsil, lymph nodes, plasmacytomas, and a renal biopsy. We were able to demonstrate multiple antigens in the same tissue and label antigens in tissues that had been stored for many years in epoxy resin. The technique allows quantitation of the gold label over plasma cell organelles and therefore gives information about the immunoglobulin secretory pathway in these cells. We found that the protein A-gold procedure compares favorably in technical ease with the immunoperoxidase, avidin-biotin peroxidase, and immunoglobulin-colloidal gold immunolabeling methods, and has added advantages in allowing precise localization and quantitation of the labeled antigen.     

Bromodeoxyuridine and lododeoxyuridine Double Immunostaining for Epoxy Resin Sections

To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.     

A physical model for simulating fusion of impaction-grafted morselized cancellous bone

This article describes a method to simulate fusion of morselized cancellous bone. The morselized cancellous bone is mixed with an epoxy adhesive, in empirically determined proportions. The mixture is then impacted into a construct. When the epoxy cures, the morselized material fuses into a cohesive, contiguous structure with a compressive modulus equivalent to that of intact cancellous bone. This model can be used to study biomechanical aspects of fused impaction grafts.     

Calcium dependence of polycystin-2 channel activity is modulated by phosphorylation at Ser812

Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser(812), a putative casein kinase II (CK2) substrate domain. Ser(812) can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo- and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca(2+) but there is a shift in this Ca(2+) dependence such that S812A is 10-fold less sensitive to Ca(2+) activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca(2+) transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser(812) modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease.     

An improved polychrome staining method for thick epoxy sections.

Improved polychrome staining of 1-1.5 mum epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial cells are stained deep blue, connective tissue pink, and fat cells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurr's (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.     

An Improved Polychrome Staining Method for Thick Epoxy Sections

Improved polychrome staining of 1-1.5 μm epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial ells are stained deep blue, connective tissue pink, and fat ells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

Genome rearrangements in host-range mutants of the polyvalent staphylococcal bacteriophage 812

Mutations extended the host range of the polyvalent bacteriophage 812 of the family Myoviridae in up to 95 % of Staphylococcus aureus strains and 43 % of strains of different coagulase-positive and -negative Staphylococcus species. Mutational changes in the genome of several host-range mutants of phage 812 were identified. Host-range mutant 812F1 harbors a deletion in endolysin gene that arose together with intron excision. Four mutants (812i, 812b, 812p, 812F3) harbor deletion in the structural gene orf8 that results from a genome rearrangement associated with intron insertion. This rearrangement was also detected in the genome of the closely related phages U16 and phi131. Another intron was discovered in the recA812 gene in these four mutants. An insertion was found in a non-coding region of the restriction fragment PstI-O of three mutants (812b, 812F3, 812g) and phages U16 and phi131. The above results contribute to the explanation of genetic factors affecting the host range of polyvalent staphylococcal bacteriophages.     

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—4 × 15 minutes, 3) absolute alcohol—3 × 40 minutes, 4) acetone—2 × 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 814,39.4 g; DMP-30, 2.6 g)— 1 hour, 6) acetone-epoxy resin 13—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.     

A Study of Criteria Permitting the Use of Plastinated Specimens for Light and Electron Microscopy

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultra-structural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.     

Preparing Epoxy Sections of Insect Tissues for Light Microscopy

Sections of aldehyde-fixed and osmium-stained insect tissues embedded in various epoxy resins were affixed to glass slides by use of a slide cover and hotplate combination. A high concentration of solvent vapor over the sections was thus maintained while they dried down on the slides, resulting in excellent flatness and adhesion. Sections were then stained at an elevated temperature with a mixture of equal parts of 3 dye solutions: 1% toluidine blue O, 1% safranin O, and saturated auramine O, all made up in 1% solution of borax in water. The method resulted in excellent differentiation of all insect tissue components including lightly chitinized structures.     

The investigation of vinylcyclohexane dioxide as a polar dehydrant for the improved retention of lipids in the Spurr embedment

The use of vinylcyclohexane dioxide (VCD) as a polar dehydrant with subsequent embedment in Spurr was studied. The utilization of Epon 812 resin (E 812), hydroxyethyl methacrylate (HEM) and hydroxypropyl methacrylate (HPM) as polar dehydrants for Epon embedment were re-examined, and a polar Epon mix was introduced. The most effective dehydration sequence was: first 90%, then 95% VCD in water for 5 min. followed by two 20 min changes of 100% VCD. After 1 hr in equal quantities of VCD and Spurr mix, tissues were infiltrated with Spurr embedment (two 1 hr changes and overnight) and finally embedded in Spurr and polymerized at 60 degrees C for 16 hr. The most utilizable polar Epon mix was determined to be Epon 812 = 50 ml, NMA=42 ml, DMP-30=1-2 ml. It was somewhat brittle but cut well with both glass and diamond knives. All four polar dehydrants were found to retain lipids and carbohydrates equally well in thin section in striated and cardiac muscle, liver, kidney and brain from the rat. The E 812 was the only dehydrant that retained lung multilamellar bodies. The possible carcinogenic effects of VCD were considered and the probably metabolism and excretion of VCD were discussed.     

RNA-Seq analysis of differentially expressed genes in rice under photooxidation

Efficient photosynthesis is critical for plant survival and growth. When plant-absorbed light exceeds the overall rate of energy conversion, it will trigger photooxidation. In this study, we selected a photooxidation mutant 812HS, it was isolated from the progeny of japonica rice (Oryza sativa L.) 812S and shows leaf yellowing and hypersensitive to photooxidation. Chloroplast ultrastructure in the leaves of 812HS showed that photooxidation resulted in significant chloroplast damage compared with 812S for changes in gene expressions in response to photooxidation stress using next-generation sequencing technologies on an Illumina HiSeq 2000 platform. A total of 88508 and 88495 genes were identified from 812S and 812HS, respectively. Expressions of 1199 genes were significantly upregulated, while 1342 genes were remarkably downregulated in 812HS. These genes were notably enriched in the 21 KEGG pathways. Based on their expression patterns, several key pathways were identified to be involved in the photooxidation of 812HS. qRT-PCR analysis further confirmed the results of RNA-Seq. This study enabled us to integrate analysis of RNA-Seq in rice and offered a deeper insight into the molecular mechanisms in response to photo-oxidative stress and provided clues for further critical gene identification in the protective mechanisms against photooxidation.     

A Simple Methylene Blue-Azure Ii-Basic Fuchsin Stain for Epoxy-Embedded Tissue Sections

One micron-thick sections of tissues fixed in glutaraldehyde, or in glutaraldehyde followed by osmium tetroxide, and embedded in a variety of plastic resins were stained in a methylene blue-azure II solution at 65 C, then counterstained in 0.05% basic fuchsin in 2.5% ethanol at room temperature (24 C). Considerable variation was found in methylene blue-azure II staining times for different embedding media. Aged Epon-812 required less staining time than freshly polymerized blocks of Epon-812. The procedure is a simple, rapid staining technique suitable for photomicrography and tissue orientation for electron microscopy.     

A polychromatic staining method for epoxy embedded tissue: a new combination of methylene blue and basic fuchsine for light microscopy

A simple and rapid method is described for staining semithin sections of material embedded in epoxy resin for observing tissues prior to transmission electron microscopy. The method is suitable for tissue fixed with a glutaraldehyde-formaldehyde mixture and postfixed in osmium tetroxide. No etching or oxidizing procedures are necessary. Sections 0.5-0.8 µm thick are dried onto a slide and stained with either 0.75% methylene blue and 0.25% azure B or 0.5% methylene blue and 0.5% azure II in 0.5% aqueous borax and heated over a flame for 8-10 sec. The slides are rinsed with water, then stained the same way with 0.1% basic fuchsine in 5% aqueous ethanol. Cytoplasm stains blue; nuclei darker blue; collagen, mucus and elastin pink to red; fat and intracellular lipid droplets gray-green.     

A polychromatic staining method for epoxy embedded tissue: a new combination of methylene blue and basic fuchsine for light microscopy

A simple and rapid method is described for staining semithin sections of material embedded in epoxy resin for observing tissues prior to transmission electron microscopy. The method is suitable for tissue fixed with a glutaraldehyde-formaldehyde mixture and postfixed in osmium tetroxide. No etching or oxidizing procedures are necessary. Sections 0.5–0.8 µm thick are dried onto a slide and stained with either 0.75% methylene blue and 0.25% azure B or 0.5% methylene blue and 0.5% azure II in 0.5% aqueous borax and heated over a flame for 8–10 sec. The slides are rinsed with water, then stained the same way with 0.1% basic fuchsine in 5% aqueous ethanol. Cytoplasm stains blue; nuclei darker blue; collagen, mucus and elastin pink to red; fat and intracellular lipid droplets gray-green.