Demonstrating the Osseous Skeleton of Human Embryos and Fetuses

Transparent human embryos and fetuses whose osseous skeletons are stained in toto by alizarin red S are successfully prepared when the KOH clearing of the soft tissues and the alizarin staining of the bones are performed simultaneously instead of independently. This modification minimizes the possibility of macerating and staining the soft tissues. Fetuses over 50 mm. CR length are skinned, eviscerated, decerebrated, defatted by dissection, fixed in 95% alcohol, bleached in H₂O₂, cleared and stained simultaneously in an aqueous solution of KOH (from 2% to 10% depending upon the size of the specimen) and .0001 to .00005% alizarin red S (solution has a pale lavender color). This solution is changed periodically to maintain the concentration of the KOH until the clearing of the tissues is complete and of the alizarin until the bones are properly stained. Tissues are dehydrated in increasing concentrations of glycerin and stored in white glycerin plus thymol.     

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.     

Differential in Toto Staining of Bone, Cartilage and Soft Tissues

The following method for staining bone and cartilage allows study of the gross cleared specimen and does not injure the tissues for subsequent microscopic study: Fix in 10% neutral formalin; bleach thoroughly in 3% H₂O₂ in sunlight. Wash in distilled water. Stain bone 24 hours in 0.01 g. of Biebrich scarlet in 100 ml. of distilled water. Destain in 95% alcohol until soft tissues and cartilage are colorless. Stain cartilage 24 hours in a pH2 buffer solution of 2.1g. of citric acid per 100 ml. of water with 0.001 g. of methylene blue. Destain in pH2 buffer solution until soft tissues are pale. Dehydrate in two changes of 95% alcohol in preparation for clearing. (This completes the destaining and may remove too much stain from the cartilage if destaining in the pH2 solution has been carried too far.) Place in Groat's clearing fluid and cover loosely so that the alcohol may evaporate, or remove the alcohol in vacuo. Groat's Mixture No. 19 is usually satisfactory.

For a combined stain, first stain bone, as above, and then apply the cartilage stain.

Seal jars with an ordinary liquid wood glue such as LePage's.     

A technique for the combination of clearing, staining, and injecting small mammals.

A combined method for clearing soft tissues, staining cartilage and bone, and injecting the vascular system of small mammals was developed using Mus musculus (house mouse). Mammalian muscle tissue remains milky or even opaque after "clearing" by previous techniques due to the relatively high content of intramuscular fat. A method employing chloroform-ethanol successfully renders soft tissues of mammalian specimens translucent without damaging or bleeding color from the latex injected in the circulatory system. Resulting specimens yield an excellent view of the skeletal system and the injected vascular system without obstruction by opaque tissues or disruption by physical removal of connective tissue.     

Toluidine Blue-Alizarin Red S Staining of Cartilage and Bone in Whole-Mount Skeletons in Vitro

Cartilage and bone of the developing skeleton can be reliably differentiated in whole-mount preparations with toluidine blue-alizarin red S staining after FAA fixation. The recommended staining procedure is based chiefly on the use of newborn white and Swiss-Webster mice, 4-9 days postnatal, but was tested also on mice and rats 3-8 wk of age. Procedure: Sacrifice, skin, eviscerate, remove body fat, and place specimens in FAA (formalin, 1; acetic acid, 1; 70% alcohol, 8) for approximately 40 min. Stain in 0.06% toluidine blue made in 70% ethyl alcohol for 48 hr at room temperature. Use 20 volumes of stain solution to the estimated volume of the specimen. Destain soft tissues in 35% ethyl alcohol, 20 hr; 50%, 28 hr; and 70%, 8 hr. Counterstain in a freshly prepared 1% aqueous solution of KOH to which is added 2-3 drops of 0.1% alizarin red S per 100 ml of solution. Each day for 3 days, transfer the specimen to a fresh 1% KOH-alizarin mixture, or until the bones have reached the desired intensity of red and soft tissues have cleared. Rinse in water, and place in a 1:1 mixture of glycerol and ethyl alcohol for 1-2 hr, then transfer the specimen to fresh glycerol-alcohol for final clearing and storage. Older mice and rats require procedural modifications: (1) fixation for 2 hr, (2) 0.12% toluidine blue, (3) maceration for 4 days in 3% KOH-alizarin, and (4) preliminary clearing for 24 hr in a mixture of glycerol, 2; 70% ethyl alcohol, 2; and benzyl alcohol, 1 (v/v) before placing in a 1:1 alcohol-glycerol mixture.     

Coupling non-destructive DNA extraction and voucher retrieval for small soft-bodied Arthropods in a high-throughput context: the example of Collembola

Here, we describe a simple method adapted for high-throughput protocols allowing voucher specimen recovery for Collembola and by extension for other soft-bodied small arthropods. A standard extraction protocol was tested to examine the effects of lysis duration (1, 2, 4, 12 h) on DNA concentration, amplification success and specimen condition. Good quality DNA was obtained after 1 h of lysis, while voucher condition was fine for up to 12 h. The lysis step substantially shortens the clearing process necessary for morphological examination.     

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.     

利用面包虫制作小兽头骨标本方法的探讨

我们利用面包虫虫蚀制作小型兽类头骨标本,取得了较好的效果.采用分组对照的方法,依据蝙蝠头骨大小、标本浸制液种类、标本浸制时间、是否剔除舌、下颌软组织和颅骨两侧大片肌肉和标本清水浸泡处理时间等5个影响因素,设计蝙蝠头骨标本制作的实验（共27只蝙蝠头骨,分7个组）.结果表明,利用面包虫虫蚀制作的头骨标本完整、干净,头骨较小的标本虫蚀时间较短,酒精或甲醛浸制一定时间的标本比新鲜标本更适合用虫蚀方法制作头骨,剔除肌肉等组织以及清水浸泡处理和适当提高实验温度均可以提高标本的制作速度,温湿度对标本制作过程有较大影响.     

A Technique for the Combination of Clearing, Staining, and Injecting Small Mammals

A combined method for clearing soft tissues, staining cartilage and bone, and injecting the vascular system of small mammals was developed using Mus musculus (house mouse). Mammalian muscle tissue remains milky or even opaque after “clearing” by previous techniques due to the relatively high content of intramuscular fat. A method employing chloroform-ethanol successfully renders soft tissues of mammalian specimens translucent without damaging or bleeding color from the latex injected in the circulatory system. Resulting specimens yield an excellent view of the skeletal system and the injected vascular system without obstruction by opaque tissues or disruption by physical removal of connective tissue.     

Analysis of effects of friction on the deformation behavior of soft tissues in unconfined compression tests

Frictionless specimen/platen contact in unconfined compression tests has traditionally been assumed in determining material properties of soft tissues via an analytical solution. In the present study, the suitability of this assumption was examined using a finite element method. The effect of the specimen/platen friction on the mechanical characteristics of soft tissues in unconfined compression was analyzed based on the published experimental data of three different materials (pigskin, pig brain, and human calcaneal fat). The soft tissues were considered to be nonlinear and viscoelastic; the friction coefficient at the contact interface between the specimens and platens was assumed to vary from 0.0 to 0.5. Our numerical simulations show that the tissue specimens are, due to the specimen/platen friction, not compressed in a uniform stress/strain state, as has been traditionally assumed in analytical analysis. The stress of the specimens obtained with the specimen/platen friction can be greater than those with the frictionless specimen/platen contact by more than 50%, even in well-controlled test conditions.     

Rapid chemical clearing of white matter in the post-mortem human brain by 1,2-hexanediol delipidation

Three-dimensional (3D) imaging based on chemical tissue clearing in the post-mortem human brain is a promising approach for stereoscopic understanding of central nervous system diseases. Especially, delipidation of lipid-rich white matter (WM) is a rate-determining step in human brain clearing by hydrophilic reagents. In this study, we described the rapid delipidation of WM by a 1,2-hexanediol (HxD)-based aqueous solution. HxD delipidation enabled rapid clearing of a formalin-fixed human brain specimen including the WM. Although harsh HxD delipidation was applied to the brain tissue, conventional pathological staining patterns and various types of antigenicity were sufficiently preserved. Furthermore, HxD delipidation was compatible with 3D imaging of fluorescently-labeled tissue samples. HxD delipidation could be useful in future 3D neuropathological diagnosis.     

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.     

High strain rate compressive properties of bovine muscle tissue determined using a split Hopkinson bar apparatus

The polymeric split Hopkinson pressure bar (PSHPB) apparatus is introduced as a means for measuring the high strain rate (1,000-2,500 s(-1)) compressive properties of soft tissues. Issues related to specimen design are discussed, and protocols are presented for specimen preparation. Proposed specimen geometries were validated using high-speed photography. Stress-strain data were obtained for high strain rate compression of bovine muscle tissue to strains as high as 80%. The stress-strain curves were found to be strain rate-sensitive and concave upward, as is typical of soft tissues. Rigor had a significant impact on the material properties between 5 and 24 h post mortem, while at longer times, properties returned essentially to their pre-rigor values. This study presents some of the first published high rate properties of muscle tissue, data that are urgently for advanced modeling of the human body and for evaluation of safety systems for the human body.     

An angiographic technique for three-dimensional determination of arterial supply patterns in cadaver soft tissue.

A method for arterial tree mapping that can be used in cadaver soft tissue is presented. In situ angiograms and photographs are supplemented with profile angiograms of relatively narrow bands of tissue from the removed specimen. The described method was better suited for mapping the course and supply patterns of a soft-tissue arterial network than either in situ angiograms or dissection. While practical problems were encountered with most of the solutions used for providing radiopacity or structural support to the vessels, pure barium sulfate was found to be suitable because it filled the vascular tree to the capillary level without leakage during excision of the specimen.     

Investigating full-field deformation of planar soft tissue under simple-shear tests

Finite simple shear test characteristics like specimen geometry and boundary conditions could affect the deformation homogeneity during the test. In order to ensure that the parameters of constitutive equations obtained from finite simple shear tests are appropriate, the deformation homogeneity of the specimen during simple-shear test should be examined. The Fourier transform moiré method (FTM) was used to examine the deformation uniformity of a porcine skin specimen in a finite simple shear test. The effects of clamping prestrain (0.15 and 0.3 engineering strain) and specimen geometry (5x5, 5x3.75, and 5x2.5cm) were investigated. These effects include in-plane deformation altered by clamping prestrain, slippage between specimen and clamps, and out-of-plane deformation. The experimental results showed that the wide specimen had more severe deformation alteration by clamping prestrain and was easier to slip out of the clamps when the shear angle is large. Furthermore, in all test configurations, the out-of-plane deformation is significant when the shear angle is large, and a narrow specimen is prone to have out-of-plane deformation. This study may provide guidelines for the selection of specimen aspect ratio and clamping prestrain when studying the material response of soft tissues under simple-shear tests.     

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.^1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light³ do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.⁴ Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.     

Whole‐body clearing of beetles by successive treatment with hydrogen peroxide and CUBIC reagents

Internal tissues of multicellular organisms cannot directly be seen because they contain pigments. For this reason, whole‐body clearing methods have been developed and applied to mammals such as mice. Insects such as beetles, however, cannot be cleared by the mammalian method because of pigments such as melanin in their exoskeletons. In this study, we tried to develop a whole‐body clearing method for large beetles. We first bleached the exoskeleton using a hydrogen peroxide treatment, and applied advanced Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) reagents to make the internal tissues transparent. The combined method of hydrogen peroxide and advanced CUBIC allowed us to successfully undertake whole‐body clearing of large beetles.     

On a class of finite deformations of elastic soft tissues

Due to the complicated physiological structure of soft biological tissues, stresses can only be measured after the specimen has been stretched to many times of its related length. Therefore, the classical constitutive equations of finite elasticity developed for vulcanized, rubbery materials and the linear theories developed for most engineering materials cannot be applied to soft tissues which are highly elastic in nature. In this article, utilizing a mechanical model developed by Demiray for soft tissues, a class of finite deformations of some tissues is studied and the results are compared with experiment and the existing literature. These problems are the simultaneous extension and twisting of a circular cylindrical bar, the bending of a rectangular block, and the pure shear of a rectangular prism. It is believed that solutions to these problems may find some applications in plastic surgery. Most of this work was done while one of the authors (H. D.) was visiting McMaster University (Hamilton, Ontario) during summer 1980 and supported through NSERC Grant 4364 and the other (M.L.) was Professor of Engn. Mechs. there.     

Conventional xylene and xylene-free methods for routine histopathological preparation of tissue sections

Abstract

Xylene customarily has been used as a clearing agent for routine tissue processing. Because xylene is a relatively hazardous solvent, laboratories are under pressure to seek less toxic alternatives for routine use. We prepared 30 paired soft tissue specimens for routine histopathological evaluation using conventional xylene and xylene-free methods to evaluate and compare their efficacy for fixation, processing, embedding, staining and turnaround time. All specimens were measured before and after processing. Three pathologists evaluated and scored the histological sections. Tissue shrinkage was greater when using the xylene method compared to the xylene-free method. The quality of tissue sections including tissue architecture; quality of staining; preservation of epithelial, fibrous, glandular, muscle and adipose tissue; inflammatory cells; and vascular tissue was better after using the xylene method, but differences were not statistically significant. Xylene-free method produced adequate results that nearly equaled the xylene method. Added advantages included cost effectiveness, better working atmosphere and decreased toxicity.