Polyethylene glycol embedment for histological studies of bean seed testa of low moisture content

Light microscopic examination of the structure of seed testa of snap and semihard bean seeds with 6% and 12% moisture contents in paraffin sections was unsuccessful because of poor paraffin infiltration and subsequent separation of subjacent and palisade cell layers. We devised an alternative method using polyethylene glycol (PEG) as the embedding material. Specimens were killed and fixed in the usual manner. They were then run up through a graded series (25, 50, 75, 100%) of PEG 1000 to PEG 1450, and finally embedded in a mixture of PEG 1450 and 4000 (19:1 by weight). Transverse and longitudinal sections retained excellent morphological detail and were suitable for histological study. Sections temporarily stained with 0.025% thionin allowed good quality photomicrographs.     

Removal of peroxides in polyethylene glycols by vacuum drying: Implications in the stability of biotech and pharmaceutical formulations

The purpose of this study was to investigate the utility of vacuum drying for removing peroxides from polyethylene glycols (PEGs). PEG solutions (PEG 1450 and PEG 20000) containing varying levels of peroxides were prepared by storing under different light and temperature conditions. PEGs containing low and high levels of peroxides were vacuum dried from dilute and concentrated solutions (2.5%, 7.5%, 15%, and 50% wt/vol of PEG 1450 and 2.5%, 7.5%, 15%, and 25% wt/vol of PEG 20000). Ferrous ion oxidation in presence of ferric ion indicator xylenol orange (FOX) colorimetric assay was used to determine the concentration of peroxides. Peroxide content in PEGs increased upon storage. The increase was more pronounced when PEGs were stored at higher temperatures and exposed to light. Vacuum drying at 0.1 mm Hg for 48 hours at 25°C resulted in greater than 90% decrease in the level of peroxides in all cases except when high peroxide containing 25% wt/vol solution of PEG 20000 or 50% wt/vol solution of PEG 1450 were dried. The reduction in the level of peroxides for PEGs dried from high peroxide containing 25% wt/vol solution of PEG 2000 and 50% wt/vol solution of PEG 1450 was found to be 88% and 52%, respectively. Oxidation of methionine in Met-Leu-Phe peptide was significantly reduced when vacuum-dried PEGs were used. Vacuum drying PEG solutions at low pressures is an effective method for the removal of the residual peroxides present in commercially available PEGs. Published: July 28, 2006     

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.     

Two-step process for initial capture of plasmid DNA and partial removal of RNA using aqueous two-phase systems

In this paper, a two-step process for initial capture of plasmid DNA (pDNA) and partial removal of RNA using polyethylene glycol (PEG) and di-potassium hydrogen phosphate aqueous two-phase systems (ATPS) has been investigated. A Kühni-type ATPS extraction column was prepared with 50 ml (12% (w/w) PEG 1450, 12% (w/w) phosphate) of stationary phase and loaded with crude mobile phase (26% (w/w) PEG 1450, 4% (w/w) phosphate and 70% (w/w) lysate) at a flow rate of 6 ml min⁻¹ at an impeller speed of 200 rpm. The experiment was terminated after 100 min, and after complete resettling of the phases, 45 ml of stationary phase was harvested. During a subsequent second extraction step contained 18% (w/w) PEG 300 and 14% (w/w) phosphate, a proportion of RNA, which was also concentrated during the column process, was removed. It was demonstrated that the recovery of pDNA in the second bottom phase was 89.4%, which was similar to the initial recovery after column extraction (92.1%).     

Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545-280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9 +/- 0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.     

原生质体融合构建葡萄酒降酸酵母的研究

对葡萄酒酵母1450(Chxs、Ampr)和酒酒球菌SD-2a(Chxr、Amps)的原生质体制备方法进行了研究,并采用PEG和Ca2 促融的方法进行了两菌株的跨界原生质体融合。利用放线菌酮 氨苄青霉素对融合子进行初筛,再经发酵试验复筛,从117株融合子中筛选出1株在酒精发酵的同时降解苹果酸能力强的融合子F-20,在连续传代10次后,其遗传性状稳定。融合子F-20的酒精发酵能力接近葡萄酒酵母1450,同时能够降解66%左右的苹果酸。     

Improved method for making high-affinity sections of soft tissue embedded in polyethylene glycol (PEG): its use in screening monoclonal antibodies

This article describes improvements in the immunohistologic technique for embedding highly hydrated embryonic tissue in polyethylene glycol 1000 (PEG)--a water-soluble wax of melting point 39 degrees C--and compares the PEG sections with frozen and polyester-wax sections. The main improvement ensures that relatively large PEG sections (8 X 3 mm) stretch out and adhere well to slides: a coat of albumen and glycerine is dried onto the slides and a fresh coat applied just before use. The embedding, sectioning, and mounting procedures, which are considerably faster than those for wax processing, have been developed for screening monoclonal antibodies against the differentiated neural crest cells in the anterior eyes of 9-day-old chick embryos. PEG sections of such eyes were a little fragile, but showed good cellular detail, similar to or better than in wax sections and considerably better than in frozen sections. The responses of PEG sections to the antibodies were far stronger than those of wax and marginally better than those of frozen sections. In one experiment using 125I-labeled rabbit anti-mouse antibody on sections previously treated with antibodies or antisera, PEG sections bound about five times as much label as wax sections and approximately 30% more than frozen sections. The main limitation of the technique is that, because of the softness of PEG, it only works well for embedding a limited range of tissues. Such PEG sections may, however, be useful for in situ hybridization as well as for immunohistochemistry.     

Staining Sulfated Mucopolysaccharides in Sections by Means of Acriflavine

Acriflavine gave insoluble salts with sulfated esters. Frozen or paraffin sections (fixed in 10% formol or Carnoy's solution) were stained in M/20 acriflavine solution and excess dye was rinsed in 95% alcohol. Then nuclei were stained with Meyer's haemalum. Thereafter the sections were washed in water, dehydrated in alcohol, cleared in xylene and mounted in balsam. Sulfated esters in the tissue sections were colored yellow or orange-yellow, generally more densely in frozen than in paraffin sections.     

PEG blocking of single pores arising on phase transitions in unmodified lipid bilayers

Changes in ionic permeability of bilayer lipid membranes (BLM) from dipalmitoyl phosphatidylcholine at temperature of phase transition in 1 M LiCl solution in the presence of polyethyleneglycols (PEG) of various molecular masses are studied. The transition of ionic membrane channels from conducting to blocked nonconducting state using polymers makes it possible to calibrate lipid pores. It is shown that low-molecular weight glycerol and PEG with molecular weights of 300 and 600 decrease the amplitude of current fluctuations through the membrane, the decrease being proportional to the size of the polymer molecule incorporated. The addition of PEG with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. The result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as PEG with molecular masses of 6000 and 20000, which are hardly incorporated in the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of PEG. It is assumed that a complete blockade of the conductivity of lipid ionic channels by PEG with molecular masses of 1450, 2000, and 3350 is due to dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic one.     

冰冻切片与石蜡切片对乳腺肿瘤的诊断价值研究

目的:分析和比较冰冻切片与石蜡切片对乳腺肿瘤的诊断价值。方法:选取480例新鲜乳腺标本,将其制成冰冻切片以及石蜡切片,根据诊断结果进行对比分析,评价乳腺肿瘤的冰冻切片与石蜡切片的对乳腺肿瘤的诊断价值。结果:经石蜡切片诊断乳腺良性肿瘤277例,占57.71%,良性肿瘤中以乳腺纤维瘤诊断居多;经石蜡切片诊断乳腺恶性肿瘤203例,占42.29%,以乳腺浸润性导管癌居多。冰冻切片诊断乳腺良性肿瘤279例,占58.13%;恶性肿瘤195例,占40.62%;延迟诊断6例,占1.25%。以石蜡切片诊断结果为金标准,冰冻切片诊断乳腺良性肿瘤的准确率为98.56%(273/277),诊断恶性肿瘤的准确率为95.07%(193/203),假阳性率为0.72%(2/277),假阴性率为2.96%(6/203),冰冻切片与石蜡切片诊断乳腺肿瘤的结果具有显著一致性,K值为0.965(P0.05)。结论:冰冻切片与石蜡切片诊断乳腺肿瘤的符合率较高,可作为术中快速病理检测的手段,但该种切片方式存在少量延迟诊断,多与术者操作经验有关,故术中应注重制片过程,提高冰冻切片质量。     

Application of cryo-compatible antibodies to human placenta paraffin sections

The hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) -fixation and paraffin embedding technique has been described to expand possibilities for immuno-labellings due to low denaturation of proteins. In this study, the issue was addressed as to whether the HOPE technique could be a useful tool in placenta tissue-based studies when only cryo-compatible antibodies are available. Such antibodies can be used on cryostat sections only, giving results of considerably inferior morphological detail as compared to routinely fixed paraffin embedded tissue sections. Commercially available, only cryo-compatible, monoclonal antibodies against a conformational epitope of HLA-G (clone MEM-G/9) and leukocyte differentiation antigens CD56, CD163 and CD34 III were selected and applied to frozen sections, routinely formalin-fixed and HOPE-fixed paraffin sections. All tested antibodies immunolocalized their antigen on cryo sections and on HOPE-fixed but not formalin-fixed paraffin sections. The HOPE technique provides an excellent preservation of protein antigenicity together with well presented morphological details in paraffin embedded placenta tissues. The detection of native or conformation-dependent epitopes in paraffin sections expands the immunolocalization possibilities in placenta research and reproductive immunology.     

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.     

56 例卵巢上皮性交界性肿瘤的临床和病理分析

目的:探讨卵巢交界性上皮肿瘤(OBT)的临床及病理特征,以供临床和病理借鉴。方法:收集病理快速冰冻切片和石蜡切片确诊的交界性上皮性卵巢肿瘤患者56例,术前均行超声检查及CA125测定。结果:56例OBT中以粘液性肿瘤最多,为33例,占58.9%,浆液性肿瘤23例占41.1%。其中以Ⅰ期多见,手术包括保守性手术治疗交界性肿瘤,预后良好。冰冻切片诊断卵巢交界性上皮肿瘤的总体准确率为64.2%。结论:诊断OBT需根据临床症状、肿瘤标记物及影像学综合判断,术中冰冻切片检查有一定帮助,但最后诊断还是应主要依据病理切片。为提高快速冰冻切片诊断的准确率,应做连续切片。     

Physical characterisations of a single-stage Kühni-type aqueous two-phase extraction column

The main parameters which influence the behaviour of phase separation in a single-stage Kühni-type aqueous two-phase extraction column containing polyethylene (PEG) and di-potassium hydrogen phosphate were characterised. Two aqueous two-phase system (ATPS) composed of 12% (w/w) PEG 1450 and 12% (w/w) di-potassium hydrogen phosphate (designated as 12/12) and 12% (w/w) PEG 1450 and 11% (w/w) di-potassium hydrogen phosphate (designated as 12/11) were chosen in this study. The hold-up ɛ_D increased with increasing impeller speeds and mobile phase flow rates. Phase separation for the 12/11 system was slower than that for the 12/12 system, which resulted in higher dispersed phase hold-up values for the 12/11 system. For 12/12 system, mass transfer of plasmid DNA (pDNA) from the dispersed mobile phase to the stationary phase increased rapidly with increasing impeller speeds of 130, 160 and 200 rpm which was reflected in the decreased values for C_T/C_To. The degree of back-mixing quantified by the axial dispersion coefficient D_ax was estimated to be 2.7 × 10⁻⁶ m² s⁻¹.     

Enthalpies of DNA melting in the presence of osmolytes

The melting of DNA in the presence of osmolytes has been studied with the intention of obtaining information about how base pair stability is affected by changes in solution conditions. In previous investigations, the melting enthalpies were assumed to be constant as osmolalities change, but no systematic evaluation of whether this condition is true has been offered. This paper presents calorimetric data on the melting of two synthetic DNA samples in the presence of a number of common osmolytes. Poly(dAdT)*poly(dTdA) and poly(dGdC)*poly(dCdG) melting have been examined by differential scanning calorimetry in solutions containing ethylene glycol, glycerol, sucrose, urea, betaine, PEG 200 and PEG 1450 at increasing osmolalities. The results show small, but significant changes in the enthalpy of melting of the two polynucleotides that are different, depending on the structure of the cosolvent. The polyols, ethylene glycol, glycerol, PEG 200 and also urea all show decreases in melting enthalpy, while betaine and sucrose display increases with increasing concentration of cosolvent. The large stabilizing PEG 1450 shows no change within the experimental errors. Using concepts relating to preferential interactions of the cosolvents with the DNA base pairs, it is possible to interpret some of the observed changes in the thermodynamic properties of melting. The results indicate that there is strong entropy-enthalpy compensation upon melting base pairs, but entropy increases dominate to cause the decreases in stability with increased cosolvent concentration. Excess hydration parameters are evaluated and their magnitudes discussed in terms of changes in cosolvent interactions with the DNA base pairs.     

Comparison of fine needle aspiration biopsy and paraffin embedded tissue sections for measuring AgNOR proteins

Abstract

Paraffin embedded tissue sections and fine needle aspiration biopsy (FNAB) are important methods for diagnosis. We compared thyroid tissue obtained by FNAB to paraffin embedded sections to determine whether there were differences in detection of the amounts of argyrophilic nucleolar organizing region (AgNOR) proteins. Twenty-two patients with papillary thyroid carcinoma were included in the study. Slides were prepared with both FNAB tissue and 3 μm sections of paraffin embedded tissue, and stained for AgNOR. One hundred nuclei per individual were evaluated; total AgNOR number/nucleus (TAn/TNn) and total AgNOR area/nuclear area (TAa/TNa) of individual cells were determined. Mean TAn/TNn and TAa/TNa values were 4.800 ± 1.118 and 13.382 ± 2.612, respectively, for FNAB samples; corresponding values were 2.406 ± 0.649 and 8.49 ± 0.893, respectively, for paraffin embedded sections. The differences between FNAB materials and paraffin embedded tissue sections were significant for the mean TAn/TNn and TAa/TNa values. Significant differences in the amounts of AgNOR protein detected were found between FNAB and paraffin embedded tissue sections.     

Lectin histochemistry of glycolipid storage diseases on frozen and paraffin-embedded tissue sections

Lectin histochemical studies were performed on frozen and paraffin-embedded brain tissue sections from six cases of galactosylceramide lipidosis (i.e., globoid cell leukodystrophy, or Krabbe's disease) in Twitcher mice and one case of canine infantile GM1-gangliosidosis. The globoid cells in Krabbe's disease stained with Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), and Bandeirea simplicifolia agglutinin-I (BS-I) in frozen sections. However, paraffin sections and frozen sections pretreated with chloroform-methanol or xylene, from the same animals, stained with Concanavlia ensiformis agglutinin (ConA), wheat germ agglutinin (WGA), and succinylated-WGA (S-WGA), in addition to staining with RCA-I, PNA, and BS-I. The affected neurons of canine infantile GM1-gangliosidosis stained only with RCA-I in frozen sections. In paraffin sections, however, these cells were negative with RCA-I but positive with BS-I, ConA, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Ulex europaeus agglutinin (UEA-I) in paraffin sections. These results indicate that in paraffin processing of glycolipid storage disease tissue, some lectin receptors are lost and others are unmasked. The retained receptors can be stained with specific lectins and could serve as markers to characterize and differentiate among the various glycolipid storage diseases.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy.

In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Detection of Mycobacterium avium subsp. paratuberculosis in formalin- fixed,paraffin embedded tissues of goats by IS900 polymerase chain reaction

The objective of the present study was to optimise and evaluate a procedure for detection of Mycobacterium avium subsp. paratuberculosis in archived formalin-fixed, paraffin embedded tissue sections from cases of naturally occurring paratuberculosis in goats. A pilot study assessed 3 procedures for extraction of DNA for detection by PCR. The procedure that gave the most consistent results involved removal of paraffin by treatment with xylene and ethanol, disruption of tissue pellets by beating with zirconium/silica beads, extraction of DNA using a DNeasy kit (Qiagen) with overnight proteinase K digestion and final ethanol precipitation. This procedure was used to analyse 82 paraffin embedded tissues (44 small intestine, 38 mesenteric lymph node) with various grades of histological lesions of paratuberculosis and acid-fast bacilli (AFB) loads. The overall sensitivity of the PCR was about 72% of all samples including both paucibacillary and multibacillary lesions. The sensitivity of the assay was 87.5% (42/48) in all paraffin sections having clearly and easily demonstrable AFB. Fifty percent of the tissue sections with rarely detectable AFB were positive by PCR. There was no significant difference (<0.05) between the sensitivity of the PCR analyses carried out on intestinal and mesenteric lymph node tissues. The results of this study suggest that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues for bacterial culture or PCR are not available due to problem of maintaining a cold chain system.     

Stability Expression of Herbicide Resistant Gene and Luciferase Gene in Calli Derived from Wheat Protoplasts

Plasmid pJIT101 containing bar gene and luc gene was transferred into wheat protoplasts mediated with PEG 1450. Transformants were then selected out in medium containing 50 mg/L Basta, a phosphinothricin (PPT)containing chemical product. The results of DNA hybridizaion indicated that both bar gene and luc gene had integerated into transformant genome. The luciferase activity has also been detected in those transformants.