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.     

A modified gold chloride technique for optimal impregnation of nerves within corneal whole mounts and dura of the albino rat

Ranvier's method of staining tissue whole mounts with gold chloride to visualize nerve fibers was modified by lengthening the incubation time in gold chloride and reducing the time in acidulated water. These simple modifications of an old technique give consistent impregnation of nerve fibers with light background staining in whole mounts of cornea and dura.     

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.     

Endothelin receptors in light-induced retinal degeneration

Excessive light exposure leads to retinal degeneration in albino animals and exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In previous studies we have described the presence of endothelin-1 (ET-1) and its receptors (ET-A and ET-B) in different sites of the mouse retina, including the retinal pigment epithelium, the outer plexiform layer (OPL), astrocytes, the ganglion cell layer (GCL), and vascular endothelia. After light-induced degeneration of photoreceptors, endothelinergic structures disappear from the OPL, but ET-1 and ET-B immunoreactivities increase in astrocytes. Here, we present novel observations about the course of light-induced retinal degeneration in BALB-c mice exposed to 1500 lux during 4 days with or without treatment with tezosentan, a mixed endothelinergic antagonist. Retinal whole mounts were immunostained with anticleaved caspase-3 (CC-3) serum to identify apoptotic photoreceptor cells within the outer nuclear layer (ONL). Glial activation was measured as glial fibrillary acidic protein (GFAP) immunoreactivity in retinal whole mounts and in Western blots from retinal extracts. Tezosentan treatment significantly reduced both the number of CC3-immunoreactive cells and GFAP levels, suggesting that inhibition of endothelinergic receptors could play a role in photoreceptor survival. Using confocal double immunofluorescence, we have observed that ET-A seems to be localized in bipolar cell dendrites, whereas ET-B is localized in horizontal cells. Our observations suggest the existence of an endothelinergic mechanism modulating synaptic transmission in the OPL. This mechanism could perhaps explain the effects of tezosentan treatment on photoreceptor survival.     

Laboratory Hints from the Literature

HERTIG, M. Hollow-ground slides for whole mounts made with the dental engine. Science, 83, 110. 1936.     

Mitotic orientation in three dimensions determined from multiple projections.

The three-dimensional orientation of mitoses in mouse small intestinal crypts of Lieberkuhn was determined from multiple projections of the mitotic figures in whole mounts of isolated intestinal crypts. We found evidence of a significant orientational bias for mitoses whose daughter cells would be added along the long axis of the crypt, and thus conform to the maintenance of the cylindrical shape of the intestinal crypt. However, we also observed many mitoses whose progeny must be rearranged if the simple cylindrical shape of the intestinal crypt is to be maintained. Our results indicate that the ultimate behavior of progeny cells and hence of local tissue form may not strictly depend on the orientation of mitosis. The methods presented may also be used in the study of mitotic orientation in other tissues.     

Stem-cell organization in mouse small intestine

We have investigated stem-cell organization in mouse small intestine (SI) by using a cellular marker induced by somatic mutation. In small intestinal whole mounts from heterozygous Dlb-1b/Dlb-1a mice stained with a peroxidase conjugate of Dolichos biflorus agglutinin (DBA-Px), mutations of Dlb-1b in stem cells result in loss of DBA-Px binding and so are recognizable as wholly or partly unstained crypts. The frequency of these clonal patterns can be measured during the accumulation of spontaneous mutations in untreated mice, or after treatment with ethylnitrosourea (ENU). The results show that there is a single infrequently dividing stem cell that maintains the epithelium of each crypt through a population of transit stem cells. The entire crypt epithelium is renewed approximately every 12 weeks.     

Factors influencing the expression of stress fibers in vascular endothelial cells in situ

The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age- matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.     

Simultaneous demonstration of adrenergic and non-adrenergic nerve fibres

Summary A technique for simultaneous demonstration of adrenergic and non-adrenergic nerve fibres is described, using methylene blue staining and fluorescence microscopy after formaldehyde treatment. The procedure is applicable to whole mounts as well as to microtome sections.     

Novel staining procedures for whole mount preparations of greater wax moth (Galleria mellonella) embryos: toluidine blue-rhodamine B,plus calcofluor white

Although hematoxylin and eosin (H & E) staining of sectioned embryonic insect material is widely used, it is time-consuming and may not provide sufficient information. We evaluated new staining procedures for embryonic whole mounts of the greater wax moth, Galleria mellonella. We compared a combination of toluidine blue and rhodamine B (TB-RB) to H & E; we also investigated calcofluor white (CFW) staining. TB-RB staining produced staining similar to H & E. TB-RB staining was less time-consuming and improved visualization of the blastoderm and its differentiation into the germ disk and serosa membrane. CFW enhanced details of mitosis in nuclei post-fertilization and stained the primary serosal membrane. Staining of whole mounts with TB-RB and CFW enabled embryonic staging that was more rapid, convenient and effective than the routine approach using H & E and fluorescent probes.     

Identification of vascular endothelial cells in murine omentum using the lectin, Dolichos biflorus agglutinin: possible applications in the study of angiogenesis

Summary The purpose of this study was to determine whether the plant lectin,Dolichos biflorus agglutinin (DBA), can be used to recognize capillary endothelial cells and their processes during angiogenesis. By means of a peroxidase conjugate of DBA, blood vessels were visualized in whole mounts and ultrathin sections of mouse omentum. A part of this mesentery normally comprises an avascular membrane that is approximately 30 µm in thickness. Changes in the vascular plexus bordering this membrane were induced by intraperitoneal injection of irradiated Landschutz cells. Vascular endothelial cells were precisely and intensely stained, and vasculogenic processes were reliably distinguished from those of other cells. This technique permitted observation of the structure and distribution of capillary sprouts, and their relationship to each other and to pre-existing blood vessels. It was discovered that filiform projections extend from sprout apices. These projections may fuse allowing adjacent sprouts to form a new capillary loop.     

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.     

The participation of alpha-actinin in the capping of cell membrane components.

By means of double fluorescence staining experiments, intracellular alpha-actinin was found to accumulate under caps and patches induced in several cells by a variety of ligands. This phenomenon was demonstrated in lymphocytes and lymphoma cells treated with anti-H-2 sera; spleen lymphocytes treated with concanavalin A or anti-immunoglobulin antibodies, and VSV-infected mouse fibroblast line MC57 treated with antiserum against viral antigens. It occurred during both rapid and slow capping processes, and could be obtained by either direct or indirect ligand-induced redistribution. These observations were carried out on whole cells. For other cytoskeletal proteins such as filamin, tropomyosin and myosin, a similar accumulation under caps was not readily apparent using whole cell mounts, although earlier experiments with frozen-sectioned cells had shown such an enrichment of myosin (as well as actin). The enrichment of alpha-actinin under the clustered surface molecules was already apparent in early stages (patching) of the capping process, with or without 10 mM sodium azide present. Prolonged incubation of the cells with the different ligands resulted in endocytosis of the ligand-receptor complex. alpha-Actinin was not associated with the inernalized complex, however, suggesting that it may dissociate from the patched or capped surface structures at some stage during endocytosis.     

Retinohypothalamic projection in the mouse: Electron microscopic and iontophoretic investigations of hypothalamic and optic centers

The problem of the direct retinohypothalamic projection in mammals (Moore, 1973) was reinvestigated in the laboratory mouse by electron microscopy and cobalt chloride-iontophoresis. The time-course of the axonal degeneration in the suprachiasmatic nucleus was studied 3, 6 and 12 h, 1, 2, 4, 6, 9 and 12 days after unilateral retinectomy. Specificity of the degenerative changes was controlled by investigation of the superficial layers of the superior colliculus. The ratio of crossed to uncrossed optic fibers could could be determined by counting degenerating structures (axons and terminals) in the optic chiasma and the ipsilateral and contralateral areas of the optic tract, the suprachiasmatic nucleus, and the superior colliculus. The number of degenerating axons in the suprachiasmatic nucleus showed a maximum one day after unilateral retinectomy and was, at all stages studied, two to three times higher in the contralateral than in the ipsilateral nuclear area. In the optic tract and in the superior colliculus the number of degenerating profiles was three times higher in the contralateral than in the ipsilateral area. Retinohypothalamic connections and crossing pattern of retinal fibers were studied light microscopically using impregnation with cobalt sulfide in whole mounts of brains. Most of the optic fibers in the laboratory mouse are crossed crossed (70-80%). A bundle of predominantly crossed optic fibers runs to the suprachiasmatic nucleus.     

Polyspermic mouse eggs can dispose of supernumerary sperm

Zona-free mouse eggs inseminated with capacitated epididymal sperm in a modified Krebs-Ringer bicarbonate medium exhibited unusual kinetics of sperm incorporation. At a sperm concentration of 10⁵ cells/ml or higher, the mean number of sperm per egg reached a maximum and then decreased with time. This decrease was correlated with the abstriction of sperm in cytoplasmic blebs which formed during or slightly after second polar body abstriction, 1.5–2.5 hr postinsemination. A correlation was apparent between the degree of polyspermy and the total number of sperm lost by this mechanism. Of 82 dispermic eggs studied, 36 underwent sperm loss by blebbing, a process that restored the monospermic condition. The sequential steps in the abstriction process are depicted in micrographs of whole mounts of fixed eggs. A sperm head or male pronucleus could be seen in isolated blebs. The prevention of bleb formation by exposure of penetrated eggs to cytochalasin B largely eliminated any difference in sperm number when the mean number of sperm per egg was compared at 2, 4, and 6 hr postinsemination. Sperm abstriction may be a novel sperm exclusion mechanism employed by mammalian eggs. Evidence is also presented that an unknown mechanism of sperm exclusion is operative in mouse eggs, since sperm loss by abstriction did not account for all sperm loss.     

Quantitation of Satellite Cell Proliferation in Vivo Using Image Analysis

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei + satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 ± 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 ± 462 μm³ of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.     

Neurons with histaminelike immunoreactivity in the segmental and stomatogastric nervous systems of the crayfishPacifastacus leniusculus and the lobsterHomarus americanus

Summary We used a polyclonal antiserum against histamine to map histaminelike immunoreactivity (HLI) in whole mounts of the segmental ganglia and stomatogastric ganglion of crayfish and lobster. Carbodiimide fixation permitted both HRP-conjugated and FITC-conjugated secondary antibodies to be used effectively to visualize HLI in these whole mounts. Two interneurons that send axons through the inferior ventricular nerve (ivn) and the stomatogastric nerve to the stomatogastric ganglion had strong HLI, both in crayfish and in lobster. These ivn interneurons were known from other evidence to be histaminergic. The neuropil of the stomatogastric ganglion in both crayfish and lobster contained brightly labeled terminals of axons that entered the ganglion from the stomatogastric nerve. No neuronal cell bodies in this ganglion had HLI. Each segmental ganglion contained at least one pair of neurons with HLI. Some neurons in the subesophageal ganglion and in each thoracic ganglion labeled very brightly. Axons of projection interneurons with strong HLI occurred in the dorsal lateral tracts of each segmental ganglion, and sent branches to the lateral neuropils and tract neuropils of each ganglion. All the labeled neurons were interneurons; no HLI was observed in peripheral nerves.     

Estrogen-activated sexual behavior in male rats

Daily injections of 100 μg estradiol benzoate activated the whole pattern of sexual behavior in castrated sexually experienced male rats. If compared to rats treated daily with 100 μg testosterone propionate, the estrogen-treated males tended to have longer latencies and more mounts and intromissions prior to ejaculation. Fifty micrograms of estradiol benzoate stimulated the display of mounts and intromissions in prepuberally castrated male rats. No peripheral effects of the estrogen treatment were noted. These results suggest that estrogen has central “androgen-like” effects, but no such effects in the periphery. Estrogen treatment (5, 50, and 200 μg/kg for 3 weeks) of intact sexually experienced male rats resulted in testicular atrophy and loss of body weight, but had no significant effects on the sexual behavior.     

A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells

The fine structure of resting and activated platelets was compared using two approaches novel to this dense cytoplasm. First, rapid lysis of platelets on carbon-coated grids was following by negative staining of the "cytoskeleton." Second, a brief, minimal fixation of platelets in plasma was coupled with partial lysis and examination of the unstained whole mounts at 200 kV. The results showed that the dense ground cytoplasm of discoid, fully resting platelets appeared granular or amorphous, and microfilaments were not observed. A coiled microtubule terminated in one, free, straight end. When any slight degree of activation occurred, microfilaments could be detected in the platelets. In fully spread specimens, the amorphous character of the resting cytoplasm was strikingly altered into an interconnected network of microfilaments. Stereo views of the whole mounts showed that dense granules, 100-250 nm in diameter, appeared as if suspended in the filament nets. The results support the view that platelet activation involves a major assembly of microfilaments from amorphous precursors. The change can only be seen convincingly when stringent precautions are taken during preparation because the platelets are very easily activated by thermal or mechanical stimuli.