Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.     

Alkaline bismuth solution as an en bloc stain for formaldehyde-glutaraldehyde potassium permanganate fixed fungal spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.     

A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

LANTHANUM STAINING OF THE SURFACE COAT OF CELLS : Its Enhancement by the Use of Fixatives Containing Alcian Blue or Cetylpyridinium Chloride

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.     

Immunoelectronmicroscopic localization of actin in ionophore-treated boar sperm

Actin in ionophore-A23187 treated boar sperm has been localized by indirect immunofluorescence and immunoelectronmicroscopy (IEM), using an anti-actin monoclonal antibody. By IEM, after en bloc staining technique and treatment with 15 nm colloidal gold-IgM complex, actin was found associated with the plasma membrane (PM) and the outer acrosomal membrane vesicles and under the PM of the equatorial segment.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

On the Number of Rays in Starfish

Multiradiate starfish evolved independently in fourteen livingfamilies. Twenty living families are strictly 5-rayed. The FIVE-PLUShypothesis is that supernumerary rays develop separately fromthe five primary rays. The ontogeny of the primary rays is proposedto be highly integrated ("en bloc" hypothesis), closely timed(synchronic hypothesis) and a developmental constraint ("tamper-proof"hypothesis). The "en bloc" hypothesis postulates that the fiveprimary rays develop as a unit. The deep structure of this unitis believed to be a 2-1-2, BA-A-BA, organization. The synchronichypothesis postulates that there is only a brief time at metamorphosisduring which the "en bloc" pathway operates. There is a pausebefore the development of supernumerary rays. The "tamper-proof"hypothesis postulates that the "en bloc" pathway has no heritablevariation and cannot be co-opted for the production of supernumeraryrays. There is diversity of timing and pattern in the developmentof supernumerary rays. Postgeneration of rays in the rudimentand intercalary regeneration of rays in the imago are independentray-producing pathways that may have been co-opted variouslyand recurrently in the multiple origins of multiradiate starfish.     

Isolation of liver lysosomes by iron loading. Ultrastructural characterization

In summary, the data demonstrate that, by the use of repeated injections of an iron sorbitol complex, it is possible to isolate a fraction highly enriched in hydrolytic enzymes (60 times over the homogenate) and in well preserved lysosomes emanating almost entirely from liver parenchymal cells. The advantage of adding fixative to the bottom of the gradient and of using en bloc staining with uranyl acetate is also demonstrated.     

En bloc cervical lipectomy for treatment of the problem neck in facial rejuvenation surgery

The treatment of cervical fat in facial aesthetic surgery has received much attention in recent years. Suction lipectomy has become a very popular technique for removing cervical fat because it is easy to perform and results in few complications. This paper describes the en bloc excision of cervical fat in conjunction with rhytidectomy. The senior author has treated 1,000 patients over 17 years using this technique with a high degree of patient satisfaction and minimal morbidity. Although suction lipectomy alone may be indicated for the younger patient, our experience suggests that the en bloc excisional technique is the treatment of choice in the older patient in whom a rhytidectomy is also indicated. In contrast with suction lipectomy, we have found that the en bloc excision of cervical fat allows for more anatomic dissection and facilitates removal of greater amounts of fat and better redraping of the cervical skin.     

A mesoscopic technique for the study of the development of the peripheral system in rat foetuses

In order to study the morphogenesis of the nervous system in the rat an acetylcholinesterase in toto method for staining nervous tissue in rat foetuses was developed. Procedure: Rat foetuses of 14-22 days are fixed "en bloc" for 24 hours in a cold sucrose-formol solution. Fixed specimens are rinsed for 2 days in cold 0.22 M sucrose in a sodiumcacodylate buffer (pH 7.2). The specimens are cut (mid-)sagittally with the aid of a razor-blade, and incubated in a medium of acetylthiocholine iodide in acetate buffer (pH 5.0). Then, dehydration in glycerine/water mixtures of increasing glycerine content follows. The specimens may be stored in pure glycerine or embedded in epoxy-resin blocks and can be studied under a binocular dissecting microscope. In using this in toto staining method both the continuity of the central and peripheral parts of the nervous system as well as details up to the level of individual perikarya and motor endplates are preserved. With this mesoscopic method the three-dimensional architecture of the peripheral nervous system and its topological relations to other structures can be studies in one specimen. The exact procedure and the results as well as a method for embedding specimens in epoxy-resin blocks for teaching purposes are described. The advantages of this mesoscopic staining method for foetuses are discussed.     

Methods for removing uranyl acetate precipitate from ultrathin sections.

Precipitate resulting from en bloc staining with uranyl acetate was removed by treating sections with 15% oxalic acid in 50% methanol for 30 minutes at 40 C. Precipitate resulting from poststaining sections with hot uranyl acetate was removed by rinsing sections in 0.25-0.50% aqueous oxalic acid for 10-15 seconds at room temperature. Rinsing sections for longer than 30 seconds removed uranyl precipitate and also destained the sections. These procedures did not damage the embedding medium or cellular detail.     

Size changes in single muscle fibers during fixation and embedding.

During fixation of single muscles fibers with glutaraldehyde, the volume of the fiber shrinks 20%, recovers in rinse and osmium tetroxide to near normal volume and shrinks 20% again when staining with uranyl acetate. This suggest that osmotic properties of membranes may not have been completely lost during fixation, post-fixation and en bloc staining. Dehydration in ethanol and propylene oxide produces a further 10% shrinkage in volume. Infiltration and embedding with Epon causes an additional 15% change in volume. This gives a total shrinkage in volume of 45% which is nearly twice that of the apparent shrinkage in the volume of the myosin lattice as determined by electron microscopy.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

Enhanced ultrastructural preservation of cartilage proteoglycans in the extended state

We investigated the preservation of proteoglycan (PG) structure in rat epiphyseal cartilage using N-N-dimethylformamide (DMF) dehydration before embedding. After aldehyde fixation, specimens with and without routine osmium post-fixation were dehydrated in graded DMF and embedded in either Spurr's resin or Lowicryl K4M resin. Standard ethanol dehydration with Spurr or Lowicryl embedding techniques resulted in the formation of condensed PGs, called matrix granules. DMF dehydration before embedding greatly improved the preservation of PG structure and resulted in an extended appearance of PGs closely resembling the fine filamentous network of cartilage tissues processed by rapid freezing and freeze-substitution. However, en bloc staining of aldehyde-fixed specimens with cationic reagents before or during DMF dehydration induced the condensation of PGs and resulted in the formation of matrix granules. These observations demonstrate that DMF, a mild dehydration agent, dramatically improves PG preservation without a harmful effect on aldehyde-fixed PG structure and can be utilized regardless of routine post-fixation.     

小麦根端分生组织细胞核中核仁通道结构的电镜观察

用常规电镜和整体银染电镜观察技术对小麦根端分生组织细胞核进行了研究。发展核仁与其周边染色质之间存在通道结构。初步分析认为,染色体NORs中的rDNA是通过该通道进入到核仁的纤维中心的。     

Endoscopic procurement of tympano-ossicular allografts: alternative to the transcranial or retroauricular technique

A tympano-ossicular tissue bank complying with European Union regulations on human allografts is feasible and critical to assure that the patient receives tissue which is safe, individually checked and prepared in a suitable environment. The transcranial procurement technique has become the standard approach to procure tympano-ossicular allografts since the 1970s because it can provide en bloc allografts. Over the last 10–20 years, en bloc allografts have been abandoned and only the malleus (hammer) is left attached to the tympanic membrane. This modification enables introduction of the transmeatal procurement technique. Transmeatal procurement using readily available nasal 0° and 30° endoscopes is a feasible alternative which avoids contact with the dura mater and is not esthetically invasive to the donor. It involves a more time-consuming procurement but avoids the need for preparation of the temporal bone plug and is therefore generally more time-efficient.     

Evolution of major histocompatibility complex by “en bloc” duplication before mammalian radiation

Duplications are an important mechanism for the emergence of genetic novelties. Reports on duplicated genes are numerous, and mechanisms for polyploidization or local gene duplication are beginning to be understood. When a local duplication is studied, searches are usually done gene-by-gene, and the size of duplicated segments is not often investigated. Therefore, we do not know if the gene in question has duplicated alone or with other genes, implying that "en bloc" duplications are poorly studied. We propose a method for identification of "en bloc" duplication using mapping, phylogenetic and statistical analyses. We show that two segments present in the major histocompatibility complex (MHC) region of human chromosome 6 have resulted from an "en bloc" duplication that took place between divergence of amniotes and methaterian/eutherian separation. These segments contain members of the same multigenic families, namely olfactory receptors genes, genes encoding proteins containing B30.2 domain, genes encoding proteins containing immunoglobulin V domain and MHC class I genes. We will discuss the fact that olfactory receptors and MHC genes have undergone positive selection, which could have helped in fixation of the surrounding genes.     

Antimicrobial resistance islands: resistance gene clusters in Salmonella chromosome and plasmids

Genes conferring simultaneous resistance to different classes of antimicrobials, confer a selective advantage to the host, particularly when those corresponding antibiotics are administered. Multiple resistance genes clustered within the same genetic locus (resistance island) can be transferred en bloc to other organisms. In this chapter we review novel multidrug resistance islands recently described in Salmonella.     

High frequency of in situ hybridization on thin plastic sections of human placenta with a cDNA probe for beta hCG

We describe two different techniques with plastic embedding in in situ hybridization histochemistry (ISHH). Their applicability was demonstrated by use of human placenta of the tenth gestational week and a tritium-labeled cDNA probe for the beta-subunit of hCG. In the first method, ISHH was performed on whole pieces of tissue (en bloc ISHH) pretreated with a weak acid solution, embedded in methacrylate, and sectioned at 3 microns for autoradiography. In the second technique, en bloc ISHH was carried out on tissue pre-treated with the weak acid and thereafter with detergent to further facilitate probe penetration. An acrylic resin was used for embedding, and section thickness was reduced to 1 microns. With both techniques, beta hCG cDNA/mRNA hybrids were localized exclusively to the syncytiotrophoblast (ST), in agreement with a previous study using sections of frozen placentas for hybridization to the same probe. However, owing to the higher resolution of the plastic sections the reliability of this localization was greatly increased. The number of autoradiographic grains over the acrylic resin 1-microns sections was found to be considerably higher than that over the methacrylate 3-microns sections. This study showed that treatment of tissue with detergent before en bloc ISHH, with subsequent embedding in acrylic resin and sectioning at 1 microns, gives high resolution in combination with a high signal-to-noise ratio after autoradiography. As the acrylic resin permits cutting of ultrathin sections, the results suggest that the technique may become useful for ISHH studies at the subcellular level.