Modifications of a Method for Preparing Retinal Wholemounts for Sectioning

A method for embedding and sectioning retinal wholemounts is described. It employs a hydrophilic embedding agent, LR White, and coverslips inert to standard embedding resins. This simple technique, which has worked successfully on both enzymatically and autoradiographically labelled material, provides a means by which retinal wholemounts can be cut nearly parallel to the plane of the retinal layers. Previously prepared retinal whole-mounts can also be successfully embedded and sectioned more than a year later by the method described.     

A simple method for embedding small specimens for photomicrography and sectioning following intracellular microiontophoresis of Lucifer Yellow CH

Summary A simple method for the rapid processing of small specimens following intracellular labelling with the fluorescent naphthalimide dye Lucifer Yellow CH is described which involves embedding in glycol methacrylate-based resin on cavity slides. The technique, which may be suitable for other intracellular and extracellular fluorescent markers, permits early fluorescence photomicrography of wholemounts and subsequent recovery of the specimens for serial sectioning and further analysis. In the present study on isolated human eccrine sweat glands, the procedure has facilitated both the identification of cells from which electrical records have been made and the determination of their dye-coupling status.     

Comparative Evaluation of Methods for Estimating Retinal Ganglion Cell Loss in Retinal Sections and Wholemounts

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a⁺, βIII-tubulin⁺ and Islet-1⁺ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG⁺ and Brn3a⁺ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin⁺ RGCs was greater than Brn3a⁺ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.     

Functional assessment of glutamate clearance mechanisms in a chronic rat glaucoma model using retinal ganglion cell calcium imaging

Using optical imaging of retinal ganglion cell (RGC) calcium dynamics in living intact retinal wholemount preparations, we tested whether RGCs in an experimental rat glaucoma model were more sensitive to exogenously applied glutamate as a result of deficient glutamate clearance mechanisms. In contrast to post-natal rat RGCs in purified cultures, in which the calcium influx induced by 200 microm NMDA and 10 microm glutamate was approximately equivalent, application of up to 500 microm glutamate did not affect calcium levels in RGCs in retinal wholemounts, even though the RGCs responded to 200 microm NMDA. Glutamate (500 microm) did elicit a RGC calcium response in retinal wholemounts when glutamate transporters were inhibited pharmacologically with DL-threo-beta-benzyloxyaspartate, confirming the presence of glutamate clearance mechanisms in this intact retina preparation. The effect of glutamate was then assessed on retinas from rats with chronically elevated intraocular pressure in one eye, produced by the injection of hypertonic saline into an episcleral vein. Application of up to 500 microm glutamate had no effect on RGC calcium levels, while millimolar concentrations of glutamate induced a calcium signal in RGCs that was indistinguishable from that in fellow control retinas. Therefore, there was no evidence for a global defect in glutamate uptake in this rat model of experimental glaucoma. Imaging glutamatergic calcium dynamics of RGCs in retinal wholemounts represents a novel methodology to probe glutamate transporter function and dysfunction in an intact CNS tissue system.     

Estrogen attenuates VEGF-initiated blood-retina barrier breakdown in male rats

17β-Estradiol has been demonstrated to protect blood-brain barrier from disruption and attenuate brain injury in various conditions. The aim of this study was to investigate the effect of 17β-estradiol on the blood-retina barrier (BRB) breakdown induced by intravitreous injection of vascular endothelial growth factor (VEGF), a significant mediator of vascular permeability. Intravitreous injection of VEGF was performed to initiate BRB breakdown in male rats with PBS in the contralateral eye as control. 2 doses of 17β-estradiol and vehicle control were given to 3 groups of rats. The integrity of the BRB was quantified by Evans blue technique and assessed by fluorescent dyes in retinal sections and wholemounts. BRB breakdown was achieved by VEGF as retinal vascular permeability was increased compared with control eyes (14.66±4.09 vs. 4.94±1.20 μl/g/h, p<0.01). Vascular permeability in the 2 groups treated with 17β-estradiol was reduced compared with control (14.66±4.09 vs. 10.26±3.67 vs. 7.37±2.22 μl/g/h, p<0.01). Rhodamine isothiocyanate (RhIC) extravasation in retinal sections and Evans blue-albumin complex leakage in retinal wholemounts were also decreased in the 2 treatment groups. These results suggest that 17β-estradiol attenuates BRB breakdown induced by VEGF in male rats, which may provide a new role of 17β-estradiol in ocular diseases.     

A computational modeling for the detection of diabetic retinopathy severity

Prolonged diabetes ultimately leads to Diabetic Retinopathy (DR) which is one of the leading causes of preventable blindness in the world. Through advanced image analysis techniques are used for abnormalities detection in retina that define and correlate the severity of DR. A thorough study is done in this area in recent past years and on the basis of these studies we have developed a computer based prediction model that is used to determine the severity of DR. To identify severity DR, we have analyzed the human eye image. We have extracted some important features from human eye image i.e. Blood Artery, Optical disc, Exudates. Based on these image and data we have designed an automated system for the determination of DR severity. This automated DR severity assessment methods can be used to predict the clinical case and conditions when young clinicians would agree or disagree with their more experienced fellow members. The algorithms described in this study may be used in clinical practice to validate or invalidate the diagnoses. Algorithms or method developed here may also be used for pooling diagnostic knowledge for serving mankind. Here we have described a computational based low cost retinal diagnostic approach which can aid an ophthalmologist to quickly diagnose the various stages of DR. This system can accept retinal images and can successfully detect any pathological condition associated with DR.     

Epoxy-Slide Embedment of Cytochemically Stained Tissues and Cultured Cells for Light and Electron Microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cost-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Epoxy-slide embedment of cytochemically stained tissues and cultured cells for light and electron microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cast-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer

This report describes a method of marking nerve cells which is approximately 100 times more sensitive than those previously available. The method depends upon intracellular injection of a new, highly fluorescent dye, Lucifer Yellow CH, which can be viewed both in living tissue and after fixation and embedding. The intense fluorescence of the dye makes injected neurons visible in cleared wholemounts, where the complex three-dimensional structure of neurons is readily apparent.Three new observations have been made with Lucifer Yellow. First, many of the invertebrate neurons studied possess an extensive and complex array of fine processes not visible with other techniques. Second, dye spreads rapidly within an injected cell. Third, dye frequently spreads from the injected cell directly to certain other cells. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily, but not exclusively, between cells known to be electrically coupled.Dye-coupling in the turtle retina revealed striking and distinctive patterns of connections. Type I horizontal cells appear to be multiply connected to each other in an extensive net. Type II horizontal cells are often connected to each other in a hexagonal array. Individual type I and type II cells, widely separated, are frequently dye-coupled; in one case, they were connected by a dyefilled axon.Dye-coupling, readily observed because of the low molecular weight and the intense fluorescence of the new dye, may serve as a general method of tracing certain functional connections by morphological means, and of studying the transfer of small molecules between cells. Preliminary results suggest that systems of dye-coupled cells are substantially more common than was previously believed.     

Nucleofection of whole murine retinas

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.     

In Situ Embedding of Cell Monolayers Cultured on Plastic Surfaces for Electron Microscopy

Various procedures suitable for routine in situ embedding of cell monolayers were tested including: (1) the use of different Epon substitutes, (2) the use of different types of plas-ticware obtained from different sources, and (3) different methods of preparing capsules for sectioning. Different resins reacted differently with different plastics and type of preparation. Merck Epon substitute bound to most of the plastics tested. Ladd Epon substitute released cleanly from all plastics tested when a suitable method of preparation was used. The results show that for routine embedding of cell monolayers it is necessary to select an appropriate Epon substitute and method of preparation of capsules for the type of plasticware used. A routine method is described, with various alternative steps which can be applied when particular difficulties are encountered.     

A quick protocol for the preparation of mouse retinal cryosections for immunohistochemistry

Immunohistochemistry (IHC) using mouse retinal cryosections is widely used to study the expression and intracellular localization of proteins in mouse retinas. Conventionally, the preparation of retinal cryosections from mice involves tissue fixation, cryoprotection, the removal of the cornea and lens, embedding and sectioning. The procedure takes 1–2 days to complete. Recently, we developed a new technique for the preparation of murine retinal cryosections by coating the sclera with a layer of Super Glue. This enables us to remove the cornea and extract the lens from the unfixed murine eye without causing the eyecup to collapse. In the present study, based on this new technique, we move a step forward to modify the conventional protocol. Unlike in the conventional protocol, in this method, we first coat the unfixed mouse eyeball on the sclera with Super Glue and then remove the cornea and lens. The eyecup is then fixed, cryoprotected and sectioned. This new protocol for the preparation of retinal cryosections reduces the time for the procedure to as little as 2 h. Importantly, the new protocol consistently improves the morphology of retinal sections as well as the image quality of IHC. Thus, this new quick protocol will be greatly beneficial to the community of visual sciences by expediting research progress and improving the results of IHC.     

THE USE OF MILLIPORE FILTERS IN ULTRASTRUCTURAL STUDIES OF CELL CULTURES AND VIRUSES

A technique is described for embedding tissue culture cells that have been adsorbed or grown on Millipore filters. The acetone used during the embedding process rendered the filters transparent so that specific areas or cells could be chosen with the aid of the light microscope. Lymphoblastoid cells processed on the filters possessed well-defined plasma membranes and microvilli which were rarely present in cells from parallel cultures that were prepared by pelleting in the centrifuge. Fibroblast cells grown on filters retained their elongated appearance, in contrast to the rounded cells in pelleted preparations. Millipore filters were also used as a means of embedding virus pellets for sectioning. Preparations containing as few as 4 x 10⁸ virus particles were suitable for study by the filter technique. Crude tissue-culture harvests of vaccinia virus and purified preparations of Rauscher murine leukemia and adeno-satellite viruses were successfully examined.     

A Simple Screening Procedure for Evaluating Central Nervous System Tissue Sections Showing Structural and Cytochemical Alterations of the Blood-Brain Barrier

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 μm and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.     

An Improved Method for Embedding Brain Tissue in Albumin-Gelatin

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose. and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 μm. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.     

An improved method for embedding brain tissue in albumin-gelatin.

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose, and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 micron. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

Filtration and agar embedding applied to fine needle aspirates for tumor diagnosis by electron microscopy. A combined technique.

OBJECTIVE: To develop a novel method for processing of fine needle aspirates subjected to electron microscopic (EM) study. STUDY DESIGN: Included 70 cases of poorly differentiated malignant tumors in which a definitive diagnosis was not possible on light microscopic (LM) examination and that thus required application of an ancillary technique such as FNA/EM, for diagnosis. We have established a novel method of processing, a technique of filtration through nylon mesh filters to eliminate red blood cells (RBCs) and necrotic debris, followed by agar well embedding to avoid loss of diagnostic material during processing without centrifugation at later steps after agar embedding, thus minimizing the time required for processing. It was successfully carried out in 70 cases. RESULTS: The combined technique was extremely effective in eliminating RBCs and necrotic debris. It also avoided further loss of valuable diagnostic material. An accurate diagnosis was rendered in 70 cases; that was not possible by LM alone. The whole procedure saves two to three hours of processing as centrifugation is not required after the agar embedding step. CONCLUSION: This technique was found to be cost- and time-effective, particularly suitable for developing countries, where financial resources are limited.     

Aerosol ot Solution—An Effective Softener of Herbarium Specimens for Anatomical Study

Dried plant parts are cut into convenient sizes and soaked in a solution containing 2.5-3.3% Aerosol OT in distilled water for 5 or more hours until well penetrated by the Aerosol. After brief washing in distilled water the material can be embedded, can be sectioned freehand or, if the nature of the material permits, with a sliding microtome, without embedding. Although it is a good wetting agent, Aerosol is chemically neutral; therefore, microchemical tests can be performed successfully on material treated with it. Refractory plant tissues embedded in paraffin can be successfully softened if one face of the block is trimmed to expose the tissue, then soaked in an Aerosol solution before sectioning.