Whole Mounts of Rabbit Lens Epithelium for Cytological Study

The eye globe is cut equatorially just posterior to the equator and the anterior part fixed in acetic-acid:akohol (1:3) for 24 hours. After washing, the lens is removed, hydrolyzed, and stained by the Feulgen technic. In the SO₂ wash solution, the lens epithelium is dissected free of the lens in one piece under a binocular microscope and laid on a cover slip, cell side down. It is flattened, after radial incisions, by absorbing water around the edge, and inverted on to a drop of glychrogel on a slide. Cell and nuclear structure are observed with the aid of phase contrast.     

Cleared Whole Mounts of Shoot Apices of Angiosperms for Topographic Study

Cleared and stained whole mounts of stem apices of two Labiates and of Phaseolus plumule giving a three-dimensional picture of the apical structure have been prepared as follows. Fix the buds in formalin-acetic acid-50% alcohol (5:5:90) for 24 hr or longer and then dissect under a binocular microscope to leave only the youngest leaves surrounding the apex. Wash for several minutes in distilled water and then clear the material in a 5% solution of sodium hydroxide at approximately 40° C for 24-48 hr. Wash thoroughly in several changes of distilled water, transfer to a solution of 1% tannic acid and 0.5% sodium salicylate for up to a minute. Wash briefly in distilled water and stain in a 1.5% solution of ferric chloride until blue-black. Wash in distilled water and dehydrate through 50%, 70%, 85%, 95% and 2 changes of absolute ethyl alcohol. If the xylem is not stained well, counter-stain for a few seconds in a 0.5% solution of safranin O in a 1:1 mixture of xylene and absolute alcohol and wash out the excess stain in the same mixture. Clear in 2 changes of xylene and place on a glass slide in thick Canada balsam. Orient with needles under low magnification and cover.     

The Use of Papain in Clearing Plant Tissues for Whole Mounts

Materials killed and fixed in FAA (formalin-acetic acid-alcohol) and similar fixatives frequently are difficult to clear for whole mounts because the denatured proteins will not become soluble in NaOH and other clearing agents. If tissues are washed for 3 days in running water, then incubated at 40 C for 5-7 days in 2% papain buffered to pH 7.2 and activated with 15 ml of .02 M Na₂S, cell contents are partly digested. Normal clearing with 5-10% NaOH followed by chloral hydrate (sat. aq.) can then effect complete solublility of cell contents and their removal. Permanent slides can be made after staining (1% safranin O in 50% alcohol for 12 hr is successful), by dehydration through alcohols, clearing in xylene, and mounting in resin.     

A New Method for Preparing Slide Mounts of Whole Bodies of Microlepidoptera

A new method is described for preparing slide mounts of whole bodies of microlepidoptera to facilitate comparative morphological studies. This method conserves traditional characters of wing pattern while revealing wing venation and other morphological structures of the denuded body. Examples of new characters revealed on slide mounts of whole bodies and photographed with a Confocal Laser Scanning Microscope are given for selected species of Gelechioidea. Also, the historical use of morphological characters for defining taxa of Lepidoptera is briefly reviewed.     

Pyridine-Silver Methods for the Study of Optic Axons in Retinal Whole Mounts

The optic fibers in the retinas of diverse species may be Selectively stained and viewed en bloc in the embryonic and adult state. Treat the eye as follows 1) 50% pyridine for at least 16 hr, 2) distilled water 3-4 hr, 3) 20% H₂O₂ until the eye is a light brown, 4) 95% ethanol overnight, 5) 1.5% AgNO₂ for 2-6 days at 37 C, 6) in water, remove the vitreous, then direct 0.25% pyrogallic acid in 1.25% formalin against the retina for 2-5 secs until the optic fibers are reduced to a coffee-copper color (1-4 minutes), 7) dissect the retina and mount flat on a glass slide, 8) ewer with glycerin, apply a coverslip and fix in place with nail polish. Variants for particular species are given.

The technique offers an advantage over Golgi and methylene blue methods which tend to stain only a small percentage of fibers and frequently do not work at the earliest stages of development.     

Whole Mounts of Small Embryos Attached Directly to Glass Slides

Suspensions of sea urchin embryos spread over the surface of glass slides were attached to the glass by a rapid coagulation of the surface with alcohol. This was done either by dipping the glass slides into absolute alcohol or by a short exposure to alcohol vapor. Thereafter the slides were immediately transferred to the fixative. A suitable procedure includes fixation with Carnoy's fluid (alcohol, chloroform, acetic acid; 6:3:1) and staining with Gomori's hematoxylin after acid hydrolysis.     

Improved Gold Chloride Procedure for Nerve Staining in Whole Mounts of Rat Corneas

The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryo-protective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at-70 C in O. C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO₄-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO₄-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO₄-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O. C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced nonspecific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohisto-chemical procedures where the reaction products would be obscured by background staining.     

Durable Whole Mounts of Sebaceous Glands Colored with Oil Blue N

Studies of the distribution, abundance, size and shape of sebaeous glands are most readily made with relatively permanent whole mounts of skin samples which are: fixed in 10% formalin, washed, stained 8-16 hours with oil blue N (2 parts sat. oil blue N in 60% isopropanol: 1 part water), washed, dissected free of obscuring connective tissue, washed, and mounted on slides in glycerol gelatin. After 2 or 3 days, the edges of the cover glass are sealed with clear lacquer.     

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card, tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.     

Dissection and Preparation of Whole Mounts of Endosperm from the Seeds of Grevillea (Proteaceae)

It is sometimes very desirable to supplement the examination of serially cut microtome sections by dissection and study of whole mounts of objects. In the case of the seeds of Grerillea the endosperm forms a curious worm-like structure, the vermiform appendage, in elucidating the nature of which microtome sections alone are inadequate. The dissection of the endosperm for demonstrating this structure is very necessary; otherwise the structure is altogether overlooked. In dissecting out the endosperm, which is easily done, either fresh seeds or previously preserved seeds may be used; the former are easier for handling and yield more satisfactory results. In the case of fresh material, an immediate killing is necessary and any one of the ordinary killing fluids may be used; Bouin's fluid was tried by the writer and was entirely satisfactory. After killing, the material is stained and dehydrated, the several stages of which are best done on the slide alone on which the material is placed. Mounting is done in any of the mounting media, and Canada balsam answers the purpose quite well.     

Staining Whole Mounts of an Insect (Tribolium confusum) Reproductive System for Succinic Dehydrogenase and nonspecific esterase

The female reproductive system of Tribolium confusum was dissected in a salt solution containing NaC1, 9 gm; KC1, 0.42 gm; and CaC1₂, 0.25 gm per liter and fixed in 1% CaC1₂-10% formol, both at 0-4 C, or air dried. Staining was by standard histochemical procedures. Selective intensity of enzyme activity was as follows: dehydrogenase was predominant in the ovarioles; esterase, in the lateral oviducts. The use of iodoacetic acid and L600 as inhibitors suppressed the dehydrogenase and nonspecific esterase activity, respectively.     

Preparation of Permanent Whole Mounts of Marine Centric Diatoms to Permit Observations of Cytoplasmic Inclusions

Marine centric diatoms are highly vacuolated plant cells which often exhibit a marked tendency to plasmolyze during fixation or other manipulatory stages required to make permanent preparations which pemit the observation of cytoplasmic inclusions. Altmann's, Schaudinn's, vom Rath's, 5% acrolein, and Allen's PFA₃ are fixatives which have been found to induce relatively little plasmolysis in the species studied. If, following fixation, the fixative is removed and desalting and dehydration are carried out in a filter assembly which allows gentle suction to make gradual changes in reagent concentration and if the cells are then placed in dilute mounting medium, only a relatively small percentage of them will be plasmolyzed. In such preparations, depending upon the fixatives employed, various cytoplasmic organelles or other inclusions will be visible in the unstained material when examined with phase contrast. Fixation with Altmann's fixative and mounting the material in Zeiss phase-contrast mounting medium (L-15) after gradual desalting and dehydration gave the best results. Standard cytochemical techniques for the detection of saccharides, protein, and lipids may be incorporated in this procedure; or, all of the steps needed for embedding the material in resins, excepting polymerization, are feasible also.     

Studies on the bacterial flora of tubificid worms

Study of the bacterial flora of the gut of the turbificid worms has shown that u. v. starilization, serial culturing, and standard bacterial identification procedures can be employed successfully. Eighteen species of bacteria were isolated and identified from the gut of tubificid worms. Eleven of the species were Gram negative which may be reflected in their possible association with chlorogonal cell metabolism in tubificid worms. Of the eighteen species identified our genera and two species are on a similar list produced by Brinkhurst & Chua in an earlier study (1969) of worms from the Great Lakes. None of the organisms identified were other than common to fresh water, organic decay processes or animal digestive systems. Their role in tubificid nutrition is now under study.Supported by a grant from the Council on Faculty Research, Eastern Illinois University.     

Silver Staining, Featuring Rapid Reduction, For Whole Mounts of Retina and Optic Pathways in Chick Embryos

Developing and established nerve fibers in the retina and in superficial tracts of the brain can be stained and viewed en bloc. The method was developed on chick embryos of 2 days of incubation to several months post-hatching but could be used on other material provided that the objects of interest were within 35 μ of the surface. Procedure: (1) Place the entire eye or head in 50% pyridine for at least 16 hr. (2) Wash well for 5-7 hr with hourly changes of distilled water or with running tap water for 4-6 hr followed by several changes of distilled water. (3) Transfer to 95% ethanol for 16-48 hr. (4) Impregnate with 1.5% AgNO₃ for 2 days at 37 C. (5) Submerge in water and, when staining the retina, remove the vitreous body and apply an aqueous solution of 0.25% pyrogallic acid in 1.25% formalin by directing a narrow stream of this reducer against the retina for 2-5 sec. Wash the eye with distilled water 30-60 sec after applying the reducer. When staining the brain, remove the meninges under water, direct the stream of reducer against the brain for 20-30 sec, and rinse the brain immediately after the nerves have stained. (6) Dissect the specimen and make temporary mounts in glycerol; or, dehydrate and clear for resin mounting. The technique stains both mature and growing axons with their growth cones and sometimes their cell bodies. The fiber patterns show best on the surfaces of the retina and brain. The stain works consistently and is suited to the study of both normal and abnormal development.     

A Method for Fixation of Fish Larvae for Morphological and Genetic Studies

Journal of Ichthyology - Three aqueous buffer solutions that make it possible to stabilize DNA and also preserve the initial body shape and morphological structures are tested for fixation and...     

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.     

A Rapid Method for Making Permanent Mounts of Nematodes

A rapid method which can be used to mount and clear nematodes and their eggs is presented. Permanent mounts of certain nematodes and parasite eggs have been prepared using a medium consisting of 56 parts of a stock solution of polyvinyl alcohol (“PVA”), 22 parts phenol and 22 parts of lactic acid. The stock solution of PVA is prepared by dissolving 15 grams of PVA in 100 ml. of distilled water. This medium can be used on material killed and fixed in 10% formalin, any concentration of alcohol, alcohol-glycerin or glycerin. Results have been very satisfactory in most instances. An accompanying plate of photographs shows some of the preparations obtained by using this method.     

High-Pressure Plastic Lamination for Flexible Mounts of Cover-Slip Preparations

A Carver laminating press has been used for the lamination of small glass cover slip preparations of smears, aspiration biopsies, organ imprints and tissue culture materials between 41/2 × 57/8 × 0.012 inch thick Vinylite plastic sheets at 10,000 lb/inch² of pressure and 400° F. The procedure described was to minimize some of the difficulties encountered in the cytological assessment of a variety of tissue and cell preparations. Lamination flattens the cells sufficiently to increase cytological detail and to reduce overlapping without sacrificing staining properties. Giemsa, Wright's methylene blue, orcein, and hematoxylin-eosin stains were used in the preparations. None of these stains were altered by the procedure. Laminated cell preparations are more suitable for making automatic and visual cell counts and for counting intracellular organoids