In Toto Staining and Preservation of Peripheral Nervous Tissue

A simple quantitative modification of the in toto staining technique for nervous networks of Sihler is described. The results are demonstrated on the innervation pattern of the hard palate of the rat. Formalin fixed hard palates of rat were macerated and bleached in an aqueous solution of 3% potassium hydroxide with a few drops of 3% hydrogen peroxide added. Thereafter, the specimens were decalcified in Sinter's solution I (1 part glacial acetic acid, 1 part pure glycerin and 6 parts 1% chloral hydrate), the process being controlled by radiography. The specimens were next stained in Sutler's solution II (1 part Ehrlich's hematoxylin, 1 part pure glycerin and 6 parts 1% chloral hydrate). After staining, the non-nervous tissues were destained in Sihler'g solution I. Destaining was checked microscopically and was stopped before the finest branches of the nerves began to fade. The specimens were then washed in a weak aqueous solution of lithium carbonate and cleared in increasing concentrations of glycerin. Good visualization of nervous structures and a deep field of observation resulted; orientation of the peripheral nerves with respect to surrounding structures was readily seen and a three-dimensional image of the nervous networks was obtained.     

Staining Paraffin Sections with Protargol 5. Chloral Hydrate Mixtures, with and Without Formamide, for Fixing Peripheral Nerves

A series of experiments was directed toward finding a means of improving fixation of mammalian glandular tissue and peripheral nerves with chloral hydrate. Specimens from cat, dog, rat, guinea pig, and man were fixed in solutions of 5-15% chloral hydrate in ethyl, methyl, and propyl alcohols, both pure and diluted with varying amounts of water. Modifiers were added, including acids, alkalies, alkaloids, amines, formamide, pyridine, piperidine, and formalin. The sectioned material was stained by the 2-hour method (AgNO₃-protargol) described previously (Davenport et al., 1939). The acidification of alcoholic chloral hydrate mixtures was deleterious to fixation but alkalinization was not. Among the modifiers, formamide was the one which showed definite improvement of fixation. A 10% solution of formamide alone in 50% ethyl alcohol gave good fixation and staining of peripheral nerve trunks, but addition of 5-7% chloral hydrate to this mixture improved staining. Treatment with 1% ammoniated alcohol after fixation and before embedding was of no value in section staining. Block stains were not tried.     

A Method for Staining Pollen Tubes in Pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsia, 6 ml; 1% aqueous aniline blue, 4 ml; 1 % orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45±2 C They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45±2 C, hydrolyzed in the clearing and softening fluid at 58±1 C for SO min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

A method for staining pollen tubes in pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsin, 6 ml; 1% aqueous aniline blue, 4 ml; 1% orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45 +/- 2 C. They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45 +/- 2 C, hydrolyzed in the clearing and softening fluid at 58 +/- 1 C for 30 min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

A Note on the Staining of the Skeleton of Cleared Specimens with Alizarin Red S

A selective, progressive method for staining the skeleton in cleared specimens, developed with rat material.

Fix in 95% alcohol for at least 48 to 96 hrs. Even longer fixation is desirable. Then place in a 1% solution of KOH until the bones are clearly visible through the surrounding tissues. Transfer directly to a dilute solution of alizarin in KOH, one part alizarin to 10,000 parts of 1% KOH. Allow the stain to act until the desired intensity is attained. Fresh stain may be added if necessary.

Complete the clearing process, (1) in Mall's solution, water 79 parts, glycerine 20 parts and KOH 1 part; (2) in increased concentrations of glycerine. Store in pure glycerine.

The success of the method depends on obtaining the proper degree of clearing before staining. If the specimen is insufficiently cleared, a general staining of all tissues usually occurs.     

一种小麦叶片气孔保卫细胞观察样品的制备方法

以小麦(Triticum aestivum)花粉植株的叶片为材料, 利用整体透明技术制备小麦叶片气孔保卫细胞的观察样品, 比较了4种透明剂的样品制备效果。结果表明, 利用整体透明技术制备小麦叶片气孔保卫细胞观察样品, 无需经过叶片撕取和刮制步骤, 样品制备方法简便且高效; 用甘油溶液、饱和水合三氯乙醛及水合三氯乙醛与甘油的混合液3种透明剂处理小麦叶片后, 在普通显微镜下均可观察到清晰的气孔保卫细胞。     

A New Method Using a Modified Mayer's Hemalum at pH 6 for Demonstrating Mucous Neck Cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

A new method using a modified Mayer's hemalum at pH 6 for demonstrating mucous neck cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

The Use of Alizarin Red S To Detect and Localize Calcium in Gametophyte Cells of Ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca²⁺ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KC1 to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca²⁺. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca²⁺ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

A mesoscopic technique for the study of the development of the peripheral system in rat foetuses

In order to study the morphogenesis of the nervous system in the rat an acetylcholinesterase in toto method for staining nervous tissue in rat foetuses was developed. Procedure: Rat foetuses of 14-22 days are fixed "en bloc" for 24 hours in a cold sucrose-formol solution. Fixed specimens are rinsed for 2 days in cold 0.22 M sucrose in a sodiumcacodylate buffer (pH 7.2). The specimens are cut (mid-)sagittally with the aid of a razor-blade, and incubated in a medium of acetylthiocholine iodide in acetate buffer (pH 5.0). Then, dehydration in glycerine/water mixtures of increasing glycerine content follows. The specimens may be stored in pure glycerine or embedded in epoxy-resin blocks and can be studied under a binocular dissecting microscope. In using this in toto staining method both the continuity of the central and peripheral parts of the nervous system as well as details up to the level of individual perikarya and motor endplates are preserved. With this mesoscopic method the three-dimensional architecture of the peripheral nervous system and its topological relations to other structures can be studies in one specimen. The exact procedure and the results as well as a method for embedding specimens in epoxy-resin blocks for teaching purposes are described. The advantages of this mesoscopic staining method for foetuses are discussed.     

The use of alizarin red S to detect and localize calcium in gametophyte cells of ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca2+ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KCl to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca2+. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca2+ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

Elimination of Fat and Protein Prior to the Selective Staining of Bone

The technic of staining skeletal systems previously described is often unsatisfactory for fetal specimens of Aves, because of the large amount of fat and protein. The writer avoids this by introducing two preliminary steps: (1) The specimen is placed in equal parts of glycerin, 95% alcohol and distilled water, and 10% aqueous pepsin (with a drop of 6N HC1 added) injected into the yoik sac, with 2-3 hours incubation at 40^oC. (2) While in 5% aqueous KOH (with a few drops of 2% H₂O₂), the fat areas are injected with cellosolve; and the specimen is left in this solution until skeletal elements become clearly visible. Staining in alizarin red S then follows.     

Notes on Technic

Fixation in 20 to 40% pyridin or in 10% chloral hydrate followed by 10 to 40% pyridin gave the most consistent staining of pericellular structures in the spinal cord of cat. Chloral hydrate perfusion and soaking followed by ammoniated alcohol (Hoff's application of Cajal's method) was uniformly successful only when pyrogallol instead of hydroquinone was used as a reducing agent. Perfusion of the animal with chloral hydrate gave a rather questionable degree of improvement over fixation by simple soaking. The difficulty in selecting a routine procedure as the “best” became apparent when no single experimental variation was outstandingly superior in all animals.     

Block Staining of Nervous Tissue with Silver: III. Pericellular end-bulbs or boutons

Fixation in 20 to 40% pyridin or in 10% chloral hydrate followed by 10 to 40% pyridin gave the most consistent staining of pericellular structures in the spinal cord of cat. Chloral hydrate perfusion and soaking followed by ammoniated alcohol (Hoff's application of Cajal's method) was uniformly successful only when pyrogallol instead of hydroquinone was used as a reducing agent. Perfusion of the animal with chloral hydrate gave a rather questionable degree of improvement over fixation by simple soaking. The difficulty in selecting a routine procedure as the “best” became apparent when no single experimental variation was outstandingly superior in all animals.     

A method for the staining of intraosseous nerve fibers using Sihler’s staining technique

Abstract

Understanding nerve fiber distribution in the jaw bone is important when performing invasive surgical treatments. Both microscopic and macroscopic anatomical techniques have been developed to study innervation. Conventional methods of removing and staining these structures, however, often alter structure and lack reproducibility of the resulting specimens. We sought to optimize Sihler’s staining technique to stain intraosseous nerves in mandibles. Four cadaver specimens were used. The best staining of intraosseous nerve fibers was achieved by using the Plank-Rychlo solution. When the Styrene monomer was used, the resulting transparency was better than that obtained with glycerin under the same conditions. No significant differences were found between Sihler's staining procedure performed according to the conventional method and the procedure in which the second decalcification step was omitted. Our results demonstrate that applying Sihler’s staining technique to bones makes them transparent and allows observation of nerves while preserving the external shape of the bone and maintaining the position of intraosseous nerve fibers. Our findings suggest our Sihler staining method for intraosseous nerve fibers can provide an intermediate resolution between macroscopic and microscopic techniques.     

A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals

A simple, rapid procedure for dual staining of cartilage and bone in rodents, particularly in late gestation, has been developed for routine use. The procedure involves rapid, complete skinning of fresh eviscerated specimens following a 30 sec immersion in a 70 C water bath. The unfixed specimen is stained in a mixture of 0.14% Alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid. Specimens are then macerated in 2% KOH, cleared and hardened in 1:1 glycerin and distilled water, and stored in pure glycerin. Rapid staining of cartilage only is done in a mixture of 0.08% Alcian blue, glacial acetic acid, and ethanol, with subsequent maceration, clearing, and hardening as in the double staining procedure. Rapid staining of bone only, concurrent with maceration of soft tissue, can be done by placing fresh, unskinned specimens in a diluted mixture of alizarin red S in 2% KOH, with subsequent clearing and hardening in 1:1 distilled water and glycerin. Good quality fetal specimens can be prepared for examination by any of these procedures in a minimum of 11/2-2 days as compared to a minimum of 4-5 days for other procedures. Double stained specimens can be examined for abnormalities of the cartilage as well as bone.     

水合氯醛、乌拉坦及其1:1 混合液在SD 大鼠麻醉中的效果比较及应用

目的:比较水合氯醛、乌拉坦及其1:1混合液在SD大鼠麻醉中的效果并进一步在大鼠模型制备的麻醉中检验其效果。方法:分别采用不同剂量的水合氯醛和乌拉坦及其1:1混合液进行麻醉实验,比较其麻醉起效时间、维持时间和死亡率,并将相同剂量的1:1混合液应用于SD大鼠模型制作时的麻醉中,比较其与非模型组之间的差异。结果:水合氯醛和乌拉坦混合液麻醉大鼠的起效时间2.5±1.5分钟,与单用水合氯醛无差异(P>0.05),比单用乌拉坦起效时间短(P<0.05);维持时间107.4±4.1分钟,比单用水合氯醛、乌拉坦长(P<0.01);麻醉死亡率比单用水合氯醛低,总死亡率比单用水合氯醛、乌拉坦低。模型组大鼠的麻醉起效时间2.9±1.6分钟,维持时间108.9±4.4分钟,零麻醉死亡率,总死亡率为2.5%;与1:1混合液非模型组的麻醉效果没有明显差异。结论:水合氯醛+乌拉坦1:1混合液麻醉效果好、起效快、死亡率极低,适合用于2小时左右的SD大鼠手术或模型制作。     

Methods of Staining Plant Tissues for Differentiating the Natural Plant Oils from Petroleum Spray Oils

Petroleum, spray oils in sections of plant tissue have been distinguished from the plant oils by staining the fresh sections in the following dye solution: To a saturated aqueous solution of Nile blue sulfate, 0.5% sulfuric acid is added and the mixture is boiled under a reflux condenser for 4 or 5 hours. It should be as nearly alkaline as possible without a change of color. A solution of 50% alcohol and 50% acetone is then saturated with oil red O. One part of the Nile blue sulfate solution is then added to two parts of the oil red O solution. Allow to settle over night and filter. Stain several hours. Rinse in water and mount in glycerin jelly. A short discussion of the merits of this method and the differentiation of the spray oils by means of indophenol blue are also given.     

水合氯醛、乌拉坦及其1：1混合液在SD大鼠麻醉中的效果比较及应用

目的：比较水合氯醛、乌拉坦及其1：1混合液在SD大鼠麻醉中的效果并进一步在大鼠模型制备的麻醉中检验其效果。方法：分别采用不同剂量的水合氯醛和乌拉坦及其1：1混合液进行麻醉实验,比较其麻醉起效时间、维持时间和死亡率,并将相同剂量的1：1混合液应用于SD大鼠模型制作时的麻醉中,比较其与非模型组之间的差异。结果：水合氯醛和乌拉坦混合液麻醉大鼠的起效时间2.5±1.5分钟,与单用水合氯醛无差异（P〉0.05）,比单用乌拉坦起效时间短（P〈0.05）;维持时间107.4±4.1分钟,比单用水合氯醛、乌拉坦长（P〈0.01）;麻醉死亡率比单用水合氯醛低,总死亡率比单用水合氯醛、乌拉坦低。模型组大鼠的麻醉起效时间2.9±1.6分钟,维持时间108.9±4.4分钟,零麻醉死亡率,总死亡率为2.5%;与1：1混合液非模型组的麻醉效果没有明显差异。结论：水合氯醛＋乌拉坦1：1混合液麻醉效果好、起效快、死亡率极低,适合用于2小时左右的SD大鼠手术或模型制作。     

Spontaneous and induced activation of rat oocytes

Ovulated rat oocytes undergo spontaneous activation during in vitro culture. After extrusion of the second polar body, they do not enter interphase but are arrested again in next metaphase-like stage (M III arrest). The present study demonstrates that puromycin and chloral hydrate can trigger transition to interphase of metaphase II and spontaneously (incompletely) activated rat oocytes. The response of oocytes to these activators depends on their stage at the time of application of a stimulus. Metaphase II oocytes enter interphase at 86.8% when treated with puromycin and in 28.7% after chloral hydrate activation. Oocytes activated with chloral hydrate at the time of spontaneously induced anaphase II enter interphase at 64.8%, but after reaching the stage of telophase II their capability to shift to interphase is again low (28.8%). Finally, M III oocytes cannot be forced to enter interphase by either chloral hydrate or puromycin treatment. This study shows that resumption of the second meiotic division and transition to interphase--the two processes that normally occur in succession as a response to oocyte activatin--can be experimentally separated.