LR White sections as slot grid support films for transmission electron microscopy

The utility of LR White sections as slot grid support films for the examination of thin resin‐embedded tissue sections by transmission electron microscopy was investigated and compared with traditional formvar‐carbon films. Throughout a variety of staining procedures, which involved the use of organic solvent, oxidizing agents, strong acid and prolonged incubation, LR White support films remained intact and the attached tissue sections remained adherent. By contrast, complete loss of formvar‐carbon support films occurred in 25% of preparations during routine staining with aqueous reagents. This loss increased to 62% following staining with either alcoholic or oxidizing and acidic stains, and to 66% following prolonged (immunohistochemical) staining. Tissue contrast, ultrastructural detail and immunohistochemical staining intensity were comparable between sections on the two types of support film. The use of LR White sections as support films for slot grids represents a quick, cheap, simple and robust alternative to traditional support films and, furthermore, requires no carbon coating     

Predictive value of p63, ki‐67, and survivin expression in oral leukoplakia: A tissue microarray study

The aim of this study was to analyze the immunohistochemical expression of survivin, ki‐67, and p63 in oral leukoplakic lesions, histopathologically differentiated into dysplastic and nondysplastic. A tissue microarray containing 57 samples of biopsies from clinically classified lesions, such as leukoplakia, was immunolabeled for survivin, ki‐67, and p63. Samples were scored for percentage of positively stained. Scores were designated as follows: low = less than 25% of positive cells; and high = more than 25% of positive cells. On performing histopathological diagnosis, 20 dysplastic lesions and 37 nondysplastic lesions were seen, in which female patients (56.1%) were predominant with an average age of 58.27 years. The study showed a high expression of 37.5% for survivin, 43.7% for ki‐67, and 88.2% for p63 in dysplastic lesions. However, there was a high expression of 16.7% for survivin, 16.7% for ki‐67, and 92% for p63 in nondysplastic lesions. There is a positive correlation of expression among the three antibodies. In the association of immunoreactivity, in both dysplastic and nondysplastic lesions, increased expression of survivin reflects on the increased expression of ki‐67, and there is an overexpression of p63. In leukoplakia, the expression of survivin associated with that of ki‐67 reinforces the assumption that all these lesions are potentially malignant, regardless of histopathology; and the overexpression of p63 may indicate carcinogenic potential. These findings may help in the treatment of patients with this type of lesion.     

CFD analysis of effects on fluid flow resistance of metallic wavy structures

Microscale wrinkles can be utilized to enhance the characteristics of products. For example, a shark skin having riblet surface can move faster underwater, minimizing fluid flow resistance. We introduce a novel approach to fabricate microscale metallic wrinkles using a soft lithography and electroforming process; we also evaluated the effects of wrinkles by computational fluid dynamics (CFD) analyses. For the generation of metallic wrinkles, a UV-curable resin, NOA68T was used to fabricate a master wrinkling pattern by mechanism of compressive forces on a skin of the weakly polymerized resin layer. The master pattern was molded and replicated as metallic wrinkles on a surface via soft lithography and electroforming processes. To understand the advantages of a wavy surface on reduction of flow resistance, we carried out two- and three-dimensional (2D and 3D) CFD analyses. The drag coefficient of a wrinkled 2D square model was decreased about 17.1 %, and a 3D real wrinkle model showed 4.9 to 7.3 % reduction of it compared to without wrinkle models. We believe that it is possible to reduce the fluid flow resistance using wavy surfaces that can easily be generated selectively or wholly on an arbitrary surface.

     

Modified Feulgen staining of DNA with aqueous solution of pinacyanol.

The paper reports on the use of a quinoline dye, pinacyanol, towards staining of acid hydrolysed DNA. The dye as an aqueous solution can be used after treatment of mammalian tissue sections in concentrated phosphoric acid at 5 degrees C for 20 min followed by hydrolysis in 6N HCl at room temperature for 15 min, for staining DNA-aldehyde molecules. It has also been observed that staining of DNA-phosphate groups is also possible in sections treated with cold concentrated phosphoric acid after selective extraction of RNA. Both in situ absorption characteristics of stained nuclei as well as in vitro absorption data of an aqueous solution of the dye have been presented. It has been suggested that staining DNA-aldehydes with pinacyanol, without any primary amino group in its molecules is due to a modified Feulgen reaction.     

Detection of DNA in mammalian tissues following removal of RNA with cold phosphoric acid.

The paper deals with the staining of nuclei in mammalian tissue sections with night blue, belonging to diphenylnaphthylmethane group and is devoid of any primary amino group in its molecules but is provided with a secondary amino group and tertiary amino groups. Staining of the DNA-phosphate groups with an aqueous solution of night blue depends upon selective removal of RNA from formalin-fixed mammalian tissues by the use of cold concentrated phosphoric acid for 20 min or 75% phosphoric acid in the cold for 2 h. Moreover, sections from which RNA has been extracted can be hydrolysed in 6N HCl at room temperature for 15 min and then can be stained with the aqueous solution of the dye. Sections of tissues after only acid hydrolysis and staining also reveal very satisfactory staining of the nuclei. Possible mechanism of staining has been suggested.     

Optimization of culture conditions for an efficient xeno‐feeder free limbal cell culture system towards ocular surface regeneration

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever‐increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno‐diseases to human system. This study was aimed to establish an efficient xeno‐feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using “interactive measurements” of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno‐feeder free epithelial equivalents towards ocular surface reconstruction. Microsc. Res. Tech. 73:1045–1052, 2010. © 2010 Wiley‐Liss, Inc.     

Immunocytological detection of salivary mucins (MUC5B) on the mucosal pellicle lining human epithelial buccal cells

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high‐pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner. Microsc. Res. Tech. 77:453–457, 2014. © 2014 Wiley Periodicals, Inc.     

Tissue integrity,costs and time associated with different agents for histological bone preparation

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin‐fixed and paraffin‐embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morsés solution (Group V). Serial, 3‐μm‐thick sections were obtained and stained with hematoxylin‐eosin to calculate the percentage of osteocyte‐occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi‐quantitative analysis to verify the adjacent muscle‐to‐bone integrity and preservation of collagen fibres. The highest percentage of osteocyte‐occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8–5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p < .001). Group IV (EDTA) had the highest integrity of muscle and collagen type I and III (P < 0.01). Demineralization with 10% acetic acid was the most effective at preserving bone tissue, while 5% EDTA was the best at maintaining collagen and adjacent muscle to bone. In conclusion, nitric acid at 5% showed the most efficient result as it balanced both time and cost as a demineralizing solution.     

Ruthenium red staining of ultrathin sections of human mast-cell granules

Ruthenium Red was used for staining glutaraldehyde-fixed ultrathin sections of mast cells embedded in Epon 812. The dye was dissolved in 0.1 M ammonia, or 0.1 N acetic acid or phosphate buffer at pH 7.4, 0.1 M at a concentration of 0.5%. The ammoniacal solution stained acid mucopolysaccharides of human mast-cell granules more specifically than did the acetic solution. The probable site of the acid mucopolysaccharides is the finely granular material within the granules.     

Oolong tea extract as a substitute for uranyl acetate in staining of ultrathin sections

In conventional transmission electron microscopy, uranyl acetate staining is used to enhance the cellular components. However, uranyl acetate is considered a radioactive material that is very toxic if ingested or inhaled and subject to restrictions in many countries. In an attempt to introduce a substitute for uranyl acetate, we evaluated oolong tea extract (OTE) for staining of ultrathin sections. Tissue sections from normal rat liver representing an ideal model organ were processed according to a routine electron microscopic fixation and embedding procedure. Serial ultrathin sections were cut and processed with either routine double electron staining or 0.2% OTE staining for 30–40 min at room temperature followed by lead citrate staining (OTE staining method). Transmission electron microscopy observations revealed that all sub‐cellular structures in hepatocytes were clearly visible with OTE staining and the quality of staining was highly compatible with those of routine double staining methods. It is suggested that OTE could be used as a non‐radioactive and hazard‐free substitute for uranyl acetate in transmission electron microscopy staining.     

Free-floating cryostat sections for immunoelectron microscopy: Bridging the gap from light to electron microscopy

Frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone for two reasons: some skin basement membrane zone proteins are labile to routine chemical fixation, and skin is not amenable to vibratome sectioning. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slide-mounting, usually limit immunohistochemical study to the light microscopy level. In the present study, we introduce use of unfixed, free-floating cryostat sections for characterization of immunolocalizations of selected skin basement membrane proteins at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy. In this regard, the method renders itself useful when results of immunolabeling experiments need to be elucidated quickly at histological and ultrastructural levels as required for diagnostic and accelerated investigative strategies.     

Orseillin BB and crystal violet, a differential staining combination for paraffin sections and water mounts of fungi.

A saturated solution of orseillin BB in 3% acetic acid followed by a 1% aqueous solution of crystal violet provides an excellent differential staining for sections of ascomycetous fructifications. The technique stains a wide variety of fungus and host cells, revealing considerable morphological and cytological detail. It is appropriate for microscope slides both of unfixed material mounted in water and of picric acid-fixed paraffin sections.     

Microwave techniques for diagnostic laboratories

Microwaves (MWs) were first introduced as a method of fixation just over 20 years ago. In recent years their use has extended far beyond that of a safe, clean, and rapid method of fixation of tissue blocks and large specimens, including brains. MWs accelerate the action of cross-linking fixatives and can greatly accelerate the various stages of tissue processing to produce a paraffin block in 30 min. An extensive range of ultrafast MW-stimulated special stains has been developed, and immunohistochemical procedures can be completed in 20 min by employing MWs. Cellular antigens are distinctly better preserved in tissues fixed by MWs than by conventional cross-linking fixatives. Also, the cytomorphology of cryostat sections irradiated in Wolman's solution is clearly improved. MWs can similarly be applied for fixation and staining of preparations for transmission and scanning electron microscopy, and they also greatly accelerate polymerisation of resins. In the current climate of cost containment, this wide range of applications makes the MW oven an invaluable addition to the diagnostic laboratory.     

Histopathological patterns of ovarian lesions: A study of 161 cases

Ovarian lesions are commonly encountered pathologies that cannot be categorized clinicoradiologically. Definite diagnosis is of great importance for therapeutic and prognostic purposes. Histopathology gives accurate diagnosis in most cases. Few cases need supportive tests like immunohistochemistry. Objective: to study the histomorphological diversity of ovarian lesions, their age and location in North of Iraq (Mosul and Duhok). Patients and methods: In the period extended from January 2008 to December 2011, 161 cases of ovarian lesions were collected from pathology departments in Azadi General Hospital “Duhok” and Al-Jamhori Teaching Hospital “Mosul”. Automated tissue processor was used for histologic study and Streptavidin-biotin method on paraffin sections was applied for immunohistochemistry. Result: There was a wide age range, most being in the third decade. The right ovaries were more common involved than the left. Histologically, 58 (36%) cases were non-neoplastic and 103 were neoplastic including 90 (55.9%) benign and 9 (5.6%) malignant tumors. The remaining 4 (2.4%) cases comprised borderline serous cystadenoma. Conclusion: Most ovarian lesions were functional non-neoplastic followed by benign neoplastic. Apart from few cases, diagnosis was merely histological without any ancillary test.     

The innovative safe fixative for histology,histopathology, and immunohistochemistry techniques: “Pilot study using shellac alcoholic solution fixative”

The concerns over health and workplace hazards of formalin fixative, joined to its cross‐linking of molecular groups that results in suboptimal immunohistochemistry, led us to search for an innovative safe fixative. Shellac is a natural material which is used as a preservative in foods and pharmaceutical industries. This study was undertaken to evaluate the fixation adequacy and staining quality of histopathological specimens fixed in the “shellac alcoholic solution” (SAS), and also to determine the validity of immunohistochemical staining of SAS‐fixed material in comparison to those fixed in formalin. Fresh samples from 26 cases from various human tissues were collected at the frozen section room of King Abdulaziz University Hospital, and fixed in SAS fixative or in neutral buffered formaldehyde (NBF) for 12, 18, 24, and 48 h, and processed for paraffin sectioning. Deparaffinized sections were stained with hematoxylin and eosin (H&E) and immunostained for different antigens. The tissues fixed in SAS for >18 h showed best staining quality of H&E comparable to NBF‐fixed tissues. Comparison of the immunohistochemical staining of different tissues yielded nearly equivalent readings with good positive nuclear staining quality in both fixatives. These findings support the fixation and preservation adequacy of SAS. Furthermore, it was concluded that the good staining quality obtained with SAS‐fixed tissues, which was more or less comparable with the quality obtained with the formalin fixed tissues, supports the validity of this new solution as a good innovative fixative. Microsc. Res. Tech. 77:385–393, 2014. © 2014 Wiley Periodicals, Inc.     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

In situ hybridization and immunofluorescence on resin‐embedded tissue to identify the components of Nissl substance

It is not clear whether the Nissl substance is present at the axon hillock. To clarify this gap in knowledge, we conducted in situ hybridization (ISH) on mouse brain tissue using 30‐μm cryostat and 1–3‐μm acrylic resin sections. Cryostat and rehydrated resin sections were exposed to digoxygenin‐labeled glutamic acid decarboxylase 1 sense and antisense riboprobes. Consecutive sections from tissue embedded in resin were subjected to the ribosomal protein L26 primary antibody to determine the distribution of the ribo/polysomes. ISH results from the antisense riboprobe in both cryostat and resin‐embedded tissue sections demonstrated an abundance of message in the neurons from the substantia nigra pars reticulate. In addition, the resin sections demonstrated hybridization signal in the axon hillock of some neurons. Immunofluorescence labeling of consecutive sections using an antibody to the most abundant ribosomal protein L26 confirmed their distribution in the cell body and the axon hillock of similar neurons. Compared with the 30‐μm cryostat sections, the most striking feature of ISH in the thinner resin (2–3 μm) sections was that there was a phenomenal improvement in the overall clarity and spatial resolution. Reexamination of the axon hillock when continuous with the cell body in cryostat sections revealed that the same message was also present, except it was overlooked initially because of overlapping cell populations in thick tissue slices. As ribosomes are a component of Nissl substance, we propose that the axon hillock, like other parts of the neuron, does contain Nissl substance. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation

Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon—a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70–90 μm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti‐neuronal β‐III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20–30 μm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.     

Fasga: A new polychromatic method for simultaneous and differential staining of plant tissues

A new method based on the simultaneous action of Safranin and Alcian Blue (or green) is reported. The main advantages of this method are its simplicity and the absence of differentiation processes. Free-hand, frozen and paraffin sections can be stained with this technique. Sections are placed in 10% sodium hypochlorite for 1–5 min, rinsed in 5% acetic acid and stained for 10 min. Sections are then washed in distilled water for 1 min, rinsed in distilled water for 5 min and mounted.     

Immunohistochemical detection and quantification of T cells in the small intestine of Isospora suis-infected piglets--influence of fixation technique and intestinal segment

Quantification of immunohistochemical results constitutes an important tool in the analysis of cells and tissue that is not readily replaced by other techniques. For reliable quantification, it is essential to consider factors such as tissue fixation and tissue sampling. We report a study on the model of the intestine of Isospora suis‐infected piglets, in which we addressed (1) whether the quantity of detectable T cells in the intestinal mucosa is the same in formalin‐, HOPE®‐, and cryo‐conserved material or whether the amounts of T cells at least correlate with one another; and (2) whether single jejunal segments differ in regard to the quantity of mucosal T cells and variability of lymphocyte infiltration. Quantification of T cells in histological sections of different parts of the jejunum of 15‐22 day old piglets infected with I. suis was performed using an anti‐CD3‐antibody and stereological point counting. Area fractions of T‐cell profiles per intestinal mucosa profile were higher in cryo‐conserved samples than in HOPE®‐ and formalin‐conserved material but no correlation between different fixations could be found. The proximal part of the jejunum contained fewer T cells compared with mid‐ and end‐jejunum. Coefficients of variation did not differ between the intestinal segments. For quantification of T cells in the gut mucosa of piglets infected with I. suis, the cryo‐conserved mid jejunum seems most suitable in cases when unbiased sampling of the complete intestine is not feasible. It is generally not possible to compare quantitative results of immunostaining in samples conserved by different methods. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.