Preparation of sand fly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies

Phlebotominae sand fly specimens were prepared for histological and physiological studies. Different fixatives were tested on sectioned and whole bodied adult females in order to obtain good fixation and provide satisfactory penetration of the embedding media. All fixed specimens were infiltrated (up to seven days under 5 C) and embedded in hydroxyethyl metacrylate. Two-three m sections were stained, mounted in Canada balsam and observed by light microscopy. Best results were achieved when whole bodied insects were double fixed in Bouin's and Carnoy's fluids (4 h/2 h) and stained in Hematoxilin/Eosin or fixed in calcium formaldehyde and stained in mercury bromophenol blue.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01–0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01-0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.     

Rapid Bleach for Melanin

Complete bleaching of melanin in strongly pigmented specimens embedded in paraffin or polystyrene, and sectioned and mounted on slides, is possible in 1-3 hr at 37 C in a solution of 20 ml of benzyl alcohol, 10 ml of acetone, 5 ml of 10% hydrogen peroxide and 4 drops of a 25% ammonia solution. The bleached tissues are well preserved and tolerate further histochemical treatments. A11 the stains and reactions tested give results identical to or better than those obtained after 24-48 hr oxidation in 10% hydrogen peroxide.     

Rapid bleach for melanin

Complete bleaching of melanin in strongly pigmented specimens embedded in paraffin or polystyrene, and sectioned and mounted on slides, is possible in 1-3 hr at 37 C in a solution of 20 ml of benzyl alcohol, 10 ml of acetone, 5 ml of 10% hydrogen peroxide and 4 drops of a 25% ammonia solution. The bleached tissues are well preserved and tolerate further histochemical treatments. All the stains and reactions tested give results identical to or better than those obtained after 24-48 hr oxidation in 10% hydrogen peroxide.     

Angioarchitecture of the esophagogastric veins in portal hypertension

Based on our previous studies on the human normal venous angioarchitecture of the esophagogastric transition segment, we performed a similar analysis in 25 postmortem specimens obtained from subjects with a diagnosis of portal hypertension. The specimens were injected with India ink or barium sulfate and sectioned for histological examination. A 'weak' zone (point of lesser resistance) was described as being the most probable site of origin for the development of varicosities in the complex venous system of the esophagogastric junction. Such a system can be considered to be a physiological hemodynamic venous blockade mechanism interposed between the portal system and the systemic veins. The 'weak' zone is made up by the scarce submucous veins (shunt veins), by the submucous confluent and final confluent trunks, and by the true perforating veins of the tunica muscularis which compose a venous unity located about 3-5.5 cm above the Z (epithelioglandular) line.     

Histological changes in bovine endometrium during the estrous cycle

Endometrial biopsy specimens were obtained from 46 normally cyclic heifers at known stages of their estrous period to show precise characteristic changes. These tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following histological changes were observed during the estrous cycle. Metrorrhagia was observed on Days 0 to 1 (estrus = Day 0). Mitoses in glandular epithelium occurred on Day 5. Basal vacuolation in the surface epithelium occurred on Days 5 to 6. Leukocyte invaded the functional layer on Day 7. Stromal mitoses were observed on Days 9 to 12. The results indicate that clincians need to be aware that histological evaluation is important for the diagnosis of endometrial function and that biopsy is useful for this purpose.     

Albumen Embedding and Individual Mounting of One or Many Mammalian Ova on Slides for Fluid Processing

For studies of ova with the light or electron microscope, as well as for autoradiographic and histochemical studies, these cells need to be sectioned. The handling of individual, often hard-to-obtain, cells through fluid processing by micropipettes is time-consuming and can easily cause damage or loss of valuable specimens. A number of interesting methods have been described for handling ova or free-floating cells. In these methods cells are commonly handled in containers with fine-mesh, wire cloth bottoms, when a number of cells are involved. Unfortunately they all require special equipment not readily or easily available (Buchanan 1965; Rinaldi et al. 1966; Izquierdo 1967; Shands 1968). In our method, egg white provides a supporting matrix for mouse ova and allows one or several specimens to be mounted on a slide.     

A Demineralization Procedure for Enzymatic Histochemical Use: A Quantitative Succinic Dehydrogenase Assay

Thinnest practical slices of bones or teeth are suspended at 4 C in a 10% solution of EDTA in 0.1 M tris buffer brought to pH 6.95 with KOH pellets. The solution is stirred moderately and continuously with a magnetic stirrer until specimens are demineralized. Unwashed specimens, taken directly from the demineralizing fluid, are frozen on a block of CO₂ ice, mounted on a tissue carrier and sectioned at 6 μ in a cryostat. Histochemical stains conducted on sections stored in a slide holder enclosed in a plastic bag in a refrigerator for as long as 2 wk were successful. Quantitative studies on the preservation of succinic dehydrogenase showed nearly all of it present in specimens demineralized for 2 days, and approximately 50% remaining in specimens demineralized for 7 days. Qualitative studies with other dehydrogenases indicate that several may be similarly affected.     

Notes and queries

A method for making permanent whole mounts of flat and round worms is described. The specimens are mounted in a drop of acid fuchsin lacto-phenol on a slide and warmed for 6 hours at 60°C. The acid fuchsin is replaced by light cotton-blue (anilin blue, W. S.) in lacto-phenol, till the desired contrast is obtained. After this, the forms are mounted in pure lacto-phenol, using the coverglass. The margin of the coverglass is sealed with the sealing media devised by Dade and Waller (equal parts of damar balsam and beeswax)     

THE EFFECT OF CHRONIC DORSAL ROOT SECTION ON THE CONCENTRATION OF FREE AMINO ACIDS IN THE RABBIT SPINAL CORD

Abstract— Amino acids may be involved in primary afferent excitatory neurotransmission in the spinal cord. To test this possibility the effect of chronic dorsal root section on amino acid levels of the rabbit spinal cord has been investigated. Dorsal roots L6-S2 were sectioned under anaesthesia. Control animals were subjected to similar surgical procedures but the dorsal roots were left intact. Electromyogram recordings taken 6 days after surgery confirmed the absence of sensory input to the lower lumbosacral cord of dorsal root sectioned animals although motor function was retained. In contrast to this control animals exhibited normal reflex activity. The spinal cord was removed from each animal and extracted in trichloracetic acid for subsequent analysts of amino acids on an autoanalyser. Sections of cord were retained for histological determination of neuronal degeneration. Comparison of amino acid levels in dorsal root sectioned and control animals revealed that the only excitatory amino acid to be significantly reduced by dorsal root section wasaspartic acid (–50 percent X although glutamic acid was also reduced (– 30 per cent). Two inhibitory amino acids, cystathionine and GABA, were also significantly depleted (– 50 and - 35 per cent). The possible involvement of these amino acids in spinal cord neurotransmission is discussed.     

Whole Mounts of Flat and Round worms for Morphological Studies

A method for making permanent whole mounts of flat and round worms is described. The specimens are mounted in a drop of acid fuchsin lacto-phenol on a slide and warmed for 6 hours at 60°C. The acid fuchsin is replaced by light cotton-blue (anilin blue, W. S.) in lacto-phenol, till the desired contrast is obtained. After this, the forms are mounted in pure lacto-phenol, using the coverglass. The margin of the coverglass is sealed with the sealing media devised by Dade and Waller (equal parts of damar balsam and beeswax)     

International Society for Biological and Environmental Repositories

Genetically altered mice are an important tool for biomedical research. Several transgenic mice have been created in which activation of the transgene results in production of β-galactosidase that can be detected by histological means. While preservation and subsequent visualization of enzyme activity in soft tissues can be complicated, it is particularly difficult in bone specimens, especially those that have been decalcified. For these studies, we examined the bones of parathyroid hormone-related peptide (PTHrP) knock-in mice in which expression of PTHrP resulted in β-galactosidase production. During the past decade, several studies have demonstrated the importance of PTHrP in bone. Thus, it is important to preserve and detect β-galactosidase enzymatic activity in bone for these studies. We demonstrate here that β-galactosidase was visualized better in slides with bone sections taken from PTHrP knock-in mice when bones were frozen and sectioned compared to bones that were embedded in plastic and sectioned using a microtome. Importantly, we were able to visualize β-galactosidase in plastic embedded bones when specimens were fixed, stained (X-gal), embedded in plastic, and then sectioned rather than being fixed, embedded in plastic, sectioned, then stained.     

Items from the Literature

Thinnest practical slices of bones or teeth are suspended at 4 C in a 10% solution of EDTA in 0.1 M tris buffer brought to pH 6.95 with KOH pellets. The solution is stirred moderately and continuously with a magnetic stirrer until specimens are demineralized. Unwashed specimens, taken directly from the demineralizing fluid, are frozen on a block of CO₂ ice, mounted on a tissue carrier and sectioned at 6 μ in a cryostat. Histochemical stains conducted on sections stored in a slide holder enclosed in a plastic bag in a refrigerator for as long as 2 wk were successful. Quantitative studies on the preservation of succinic dehydrogenase showed nearly all of it present in specimens demineralized for 2 days, and approximately 50% remaining in specimens demineralized for 7 days. Qualitative studies with other dehydrogenases indicate that several may be similarly affected.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

Demonstration of Cholinesterase in Teeth

Human teeth have been studied by treatment with copper thio-choline, the method developed by Koelle for demonstrating activity of both specific and nonspecific cholinesterases. Freshly extracted teeth were collected and immediately sectioned on a cutting machine designed for calcified tissues. One series of teeth was sectioned sufficiently thin for microscopic study. Another series of teeth was bisected to expose the pulp chambers to the reagents. These teeth were divided into 5 experimental groups. The first group was treated with 10^-6M di-isopropylfluorophosphate (DFP) for 30 min at 37°C and then incubated with acetylthiocholine (AThCh) for 16 to 20 hr at 37°C in order to reveal the sites of activity of the specific enzyme, AChEase. The second group was incubated in a substrate of butyrylthiocholine (BuThCh) for 12 to 16 hr at 37°C to indicate the sites of the nonspecific ChEase. The third group was incubated in AThCh for 16 to 20 hr at 37°C without previous treatment by an inhibitor in order to reveal the presence and location of both the specific and nonspecific ChEase. The fourth and fifth groups were utilized as controls. Group 4 tissues were incubated without a substrate while those of group 5 were treated with DFP and then incubated with BuThCh. The specimens then were treated with ammonium sulfide to outline the sites of ChEase activity. The thin sections were mounted directly but the series of halved teeth were decalcified, embedded in paraffin, sectioned and then mounted. By these methods the presence and location of both specific and nonspecific ChEase activity were observed in human teeth. Concentration of specific ChEase was observed along the coronal aspect of the pulp chamber and along the course of the pulpal nerves. The nonspecific ChEase was observed throughout the pulpal tissue and appeared to be concentrated along the nerves and blood vessels. Neither series of control tissues exhibited any staining in the pulp tissue.     

A Double-Embedding Technique for Thin Tissue Membranes

A new double-embedding technique for thin tissue membranes is presented. This technique is useful for thin membranes such as mesenteric membranes from rodents, which usually measure only 10 μm in thickness. Several membranes are fixed and mounted on four needles located at the bottom of a plastic box. The box is filled with agarose at 50 C and then allowed to solidify. The agarose block is then removed, dehydrated in alcohol, cleared with Histo Petrol (isoparaffin hydrocarbons), permeated with paraffin and sectioned. The morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used and makes immunohistochemical studies easier.     

The Demonstration of a new nerve-cell Organoid

A method is described for the demonstration of a new nerve-cell organoid— the binary spheroid systems (Baker bodies). Zenker-formol or acetic-osmium-bichromate materials are postchromed at 37°C. and embedded in paraffin, sectioned and mounted in the ordinary manner. The sections are colored in a 70% alcohol solution of Sudan black B, rinsed in 70% alcohol, counter-stained in Mayer's carmalum and mounted in Farrant's medium. After examination the cover may be removed and the Sudan black extracted in 96% alcohol. The sections can then be restained by the azan method of Heidenhain, and other cytoplasmic inclusions can be correlated accurately to the functional states of the spheroid systems.     

Polystyrene Embedding: A New Method for Light and Electron Microscopy

Polystyrene embedments of histological specimens can be Obtained with a solution 1 : 4 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue. which are then glued to a Plexiglas support Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections, with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy dons on a slide heated on a hot plate to 80 C; those can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fired for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron-microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and give acceptable results.     

Novel Fixation of Plant Tissue, Staining through Paraffin with Alcian Blue and Hematoxylin, and Improved Slide Preparation

Onion (Allium cepa) root tips were fixed in a proprietary solution without aldehyde, toxic metals or acetic acid. Fixed specimens were embedded in paraffin, sectioned on a rotary microtome and mounted on detergent-washed slides without adhesive. Slides with ribbon segments affixed were immersed in 0.2% aqueous alcian blue 8GX in screw-capped Coplin jars in a water bath at 50 C for 1 hr. Excess alcian blue was rinsed off under cold running tap water and the slides were immersed in quick-mixed hematoxylin at room temperature for 15 min. Stained slides were deparaffinized, rinsed with isopropanol, air dried, and coverslips were affixed with resin. Thus, the traditional paraffin microtechnique has been modified at all steps from fixation to finishing slides with coverslips.