Benzyl Alcohol/benzyl Benzoate Research Articles

Collagen crosslinking-induced corneal morphological changes: a three-dimensional light sheet Microscopy-based evaluation.

Stiffness-related eye diseases such as keratoconus require comprehensive visualization of the complex morphological matrix changes. The aim of this study was to use three-dimensional (3D) light sheet fluorescence microscopy (LSFM) to analyze unlabeled corneal tissue samples, qualitatively visualizing changes in corneal stiffness. Isolated porcine corneal tissue samples were treated with either NaCl or 0.1% glutaraldehyde (GTA) prior to clearing with benzyl alcohol/benzyl benzoate (BABB) and subsequently scanned with LSFM. After analysis of the LSFM data sets, the samples were embedded in paraffin to validate the results by conventional planar microscopy. In the unlabeled corneal tissue samples the 2D/3D morphology of the entire tissue volume was identified by specific autofluorescence signals. An enhancement of collagen crosslinking was induced by applying GTA to the corneal tissue. Subsequent LSFM scans showed specific morphological changes due to altered autofluorescence signals of the corneal stroma, which were confirmed by conventional histology. Therefore, LSFM analysis of corneal tissue samples allowed label-free 3D autofluorescence assessment of the corneal morphology in its anatomical context. It provides the technical basis for the examination of the pathologically altered cornea and facilitates ophthalmologic examinations of corneal diseases based on the altered tissue stiffness.

Data retrieval from archival renal biopsies using nonlinear microscopy.

Thorough examination of renal biopsies may improve understanding of renal disease. Imaging of renal biopsies with fluorescence nonlinear microscopy (NLM) and optical clearing enables three-dimensional (3D) visualization of pathology without microtome sectioning. Archival renal paraffin blocks from 12 patients were deparaffinized and stained with Hoechst and Eosin for fluorescent nuclear and cytoplasmic/stromal contrast, then optically cleared using benzyl alcohol benzyl benzoate (BABB). NLM images of entire biopsy fragments (thickness range 88-660 μm) were acquired using NLM with fluorescent signals mapped to an H&E color scale. Cysts, glomeruli, exudative lesions, and Kimmelstiel-Wilson nodules were segmented in 3D and their volumes, diameters, and percent composition could be obtained. The glomerular count on 3D NLM volumes was high indicating that archival blocks could be a vast tissue resource to enable larger-scale retrospective studies. Rapid optical clearing and NLM imaging enables more thorough biopsy examination and is a promising technique for analysis of archival paraffin blocks.

3D Optical Reconstruction of the Nervous System of the Whole-Body Marine Invertebrates.

Optical clearing of invertebrates, the number of species of which is 20 times greater than that of vertebrates, is of fundamental and applied interest for neuroscience in general. Herein, the optical clearing of invertebrates to identify their morphology and neurostructure remains unrealized as of yet. Here, we report on fast (from a few seconds to minutes) and uniform whole-body optical clearing of invertebrates (bivalves, nemertines, annelids, and anomura) of any age and thickness (up to 2 cm) possessing complicated structures and integuments. We developed the protocol unifying dimethyl sulfoxide (DMSO)-based immunostaining of the animals followed by their optical clearing with benzyl alcohol/benzyl benzoate (BABB). Confocal microspectroscopy revealed that the protocol provides an increase of the fluorescence signal by 2 orders of magnitude and decrease of the light scattering by 2 orders of magnitude, thereby accelerating the confocal bioimaging of the whole body. Moreover, by tracking the optical clearing over time with 0.3 s resolution, we revealed that the clearing process is described by the Gompertz growth function, allowing us to determine the physical mechanism of the clearing and its optical parameters. Thereby, we were able to identify in detail and to describe previously unknown neurostructures of different invertebrate animals, paving the way to discovery in neuroscience.

Tissue clearing may alter emission and absorption properties of common fluorophores

In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis.

Clearing-induced tisssue shrinkage: A novel observation of a thickness size effect.

The use of clearing agents has provided new insights in various fields of medical research (developmental biology, neurology) by enabling examination of tissue architecture in 3D. One of the challenges is that clearing agents induce tissue shrinkage and the shrinkage rates reported in the literature are incoherent. Here, we report that for a classical clearing agent, benzyl-alcohol benzyl-benzoate (BABB), the shrinkage decreases significantly with increasing sample size, and present an analytical formula describing this.

An easy and rapid staining method for confocal microscopic observation and reconstruction of three-dimensional images of echinoderm larvae and juveniles.

The morphologies of the internal organs of echinoderm larvae and juveniles are difficult to study using conventional optical microscopes because of their structural complexity and opaqueness. This paper describes an easy and rapid protocol involving Nile blue staining followed by benzyl alcohol/benzyl benzoate (BABB) clearing to overcome this limitation. This method was developed for a three-dimensional (3D) analysis of the internal structures of advanced larvae and juveniles of echinoderms (the sea lily Metacrinus rotundus, the sea urchin Hemicentrotus pulcherrimus, and the sand dollar Scaphechinus mirabilis) and is suitable for obtaining serial optical images by confocal microscopy without the use of specific antibodies or special reagents for labeling. Nile blue is an easy-to-use stain that offers several advantages for confocal microscopy such as it can stain various tissues with strong fluorescent signals without substantial bleaching during observation. We found that the strong fluorescence signal of Nile blue quickly yielded clear high-resolution optical section images for 3D reconstruction. BABB clearing rendered opaque larvae highly transparent. The clearing procedure was also easy and quick. During the process, agarose embedding prior to staining and clearing was found to be critical for handling the samples of less than 500-μm length and stabilizing their orientations. To conclude, the protocol described is useful for performing a rapid and accurate 3D morphological analysis of echinoderm larvae and juveniles.

A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II

Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we present a straightforward method for immunostaining, clearing and imaging of whole murine peripheral lymph nodes (PLNs) for detailed analysis of their architecture and discuss all procedures in detail in a step-by-step approach. Given the importance of tumor necrosis factor receptor (TNFR) signaling in development of PLNs we used TNFRI-/- and TNFRII-/- mice models as proof-of-concept for this technique by visualizing and analyzing structural changes in PLN B cell clusters and high endothelial venules (HEVs). Samples were subjected to de- and rehydration with methanol, labeled with antibodies for B cells, T cells and high endothelial venules (HEVs) and optically cleared using benzyl alcohol-benzyl benzoate. Imaging was done using LaVision light sheet microscope and analysis with Imaris software. Using these techniques, we confirmed previous findings that TNFRI signaling is essential for formation of individual B cell clusters. In addition, Our data suggest that TNFRII signaling is also to some extent involved in this process as TNFRII-/- PLNs had a B cell cluster morphology reminiscent of TNFRI-/- PLNs. Moreover, visualization and objective quantification of the complete PLN high endothelial vasculature unveiled reduced volume, length and branching points of HEVs in TNFRI-/- PLNs, revealing an earlier unrecognized contribution of TNFRI signaling in HEV morphology. Together, these results underline the potential of whole mount tissue staining and advanced imaging techniques to unravel even subtle changes in lymphoid tissue architecture.

Development of an Optimized Clearing Protocol to Examine Adipocyte Subpopulations in White Adipose Tissue.

Organic solvent dibenzyl ether (DBE)-based protocols have been widely used in adipose tissue clearing. However, benzyl alcohol/benzyl benzoate (BABB)-based clearing has been shown to offer better transparency in other tissues. The addition of diphenyl ether (DPE) to BABB (BABB-D4) is often included to preserve fluorescent signals, but its effects on adipose tissue transparency and shrinkage have not been explored. Distinct adipocyte subpopulations contribute to its cellular composition and biological activity. Here, we compared clearing solvents to create an optimized clearing methodology for the study of adipocyte subpopulations. Adipose tissues were cleared with BABB, BABB-D4, and DBE, and post-clearing transparency and tissue shrinkage were measured. An optimized protocol, including BABB-D4 clearing, delipidation, and extensive immunofluorescence blocking steps, was created to examine the spatial distribution of Wt-1 positive progenitor-derived (Type-1) adipocytes in intact mesenteric fat. Both BABB and BABB-D4 lead to significantly increased tissue transparency with reduced tissue shrinkage compared to DBE-cleared adipose tissue. Type-1 adipocytes are found in a clustered distribution with predominant residence in fat associated with the ileum and colon. This paper details an optimized clearing methodology for adipose tissue with increased tissue transparency and reduced shrinkage, and therefore will be a useful tool for investigating adipose tissue biology.

Source of Early Regenerating Axons in Lamprey Spinal Cord Revealed by Wholemount Optical Clearing with BABB.

Many studies of axon regeneration in the lamprey focus on 18 pairs of large identified reticulospinal (RS) neurons, whose regenerative abilities have been individually quantified. Their axons retract during the first 2 weeks after transection (TX), and many grow back to the site of injury by 4 weeks. However, locomotor movements begin before 4 weeks and the lesion is invaded by axons as early as 2 weeks post-TX. The origins of these early regenerating axons are unknown. Their identification could be facilitated by studies in central nervous system (CNS) wholemounts, particularly if spatial resolution and examination by confocal microscopy were not limited by light scattering. We have used benzyl alcohol/benzyl benzoate (BABB) clearing to enhance the resolution of neuronal perikarya and regenerated axons by confocal microscopy in lamprey CNS wholemounts, and to assess axon regeneration by retrograde and anterograde labeling with fluorescent dye applied to a second TX caudal or rostral to the original lesion, respectively. We found that over 50% of the early regenerating axons belonged to small neurons in the brainstem. Some propriospinal neurons located close to the TX also contributed to early regeneration. The number of early regenerating propriospinal neurons decreased with distance from the original lesion. Descending axons from the brainstem were labeled anterogradely by application of tracer to a second TX close to the spinal–medullary junction. This limited contamination of the data by regenerating spinal axons whose cell bodies are located rostral or caudal to the TX and confirmed the regeneration of many small RS axons as early as 2 weeks post-TX. Compared with the behavior of axotomized giant axons, the early regenerating axons were of small caliber and showed little retraction, probably because they resealed rapidly after injury.

Benzyl Alcohol-Benzyl Benzoate Clearing Reveals the Dose-Dependent Effect of Cyclophosphamide on Follicle Damage in Mice

Objective: Cyclophosphamide (CTX), which is commonly used in clinical chemotherapy, has a damaging effect on ovarian follicles. This study aimed to establish a new method to count the number of follicles in mouse ovaries using benzyl alcohol–benzyl benzoate (BABB)-based tissue-clearing technology and evaluate the follicle-damaging effects of different doses of CTX. Methods: C57BL/6 mice were divided into four groups and administered intraperitoneal injections of 0, 40, 80, or 120 mg/kg CTX. The serum levels of estradiol (E2) and follicle-stimulating hormone (FSH) were detected using an ELISA kit. Mouse ovaries were subjected to BABB clearing and labeled with DAPI, β-actin, and DDX4 to observe the ovarian structure and follicles. A three-dimensional software, Imaris, was used to reconstruct the ovarian structure and automatically count the number of follicles. The effects of different CTX doses on the total follicle number and estrous cycle were determined. Results: As the CTX dose increased, E2 levels in CTX mice declined from 212.3 to 57.7 pg/mL; the FSH levels increased from 3.2 to 29 ng/mL. Mouse ovaries became transparent after BABB treatment. After fixation, microscopy, and Imaris processing, immunofluorescence signals of β-actin and DAPI from all levels in intact ovaries could be obtained and follicle number in half ovaries could be automatically counted using anti-DDX4 antibody labeling. In the NC, CTX40, CTX80, and CTX120 groups, the proportion of mice in the diestrus phase was 26.67%, 51.67%, 73.33%, and 95.00%, respectively, and the total follicle number was 2,603, 1,761, 1,043, and 262, respectively. E2 levels were positively correlated with follicle number and FSH levels were negatively correlated with follicle number, indicating that the damaging effect of CTX on follicle number may be dose dependent. Conclusion: BABB can be used to clear intact ovaries from adult mice, and follicle number in half ovaries can be automatically counted. The damaging effect of CTX on follicles and the endocrine system is dose dependent.

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction.

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Neutrophils as an important pathophysiologic cellular component in the onset of AMD

AbstractVision loss from age‐related macular degeneration (AMD) is an expanding, major unmet problem due to the aging population worldwide. Inflammation has emerged as a potential cause of AMD, but how inflammation contributes to vision loss in AMD remains controversial despite intensive study around the world. Herein, we identify inflammatory signals that drive neutrophil infiltration into the retinas of early AMD patients and the Cryba1 conditional knockout mouse model that mimics an early AMD‐like phenotype. Specifically, we observed increased levels of IFNλ in early AMD that triggered neutrophil activation and lipocalin‐2 (LCN‐2) upregulation in these cells. NOD‐SCID immune‐deficient mice were injected intravenously with IFNλ activated bone marrow‐derived normal neutrophils labeled with red CMTPX dye. Ribbon‐scanning confocal microscopy (RSCM) of benzyl alcohol benzyl benzoate (BABB) cleared eyes, showed homing of neutrophils into the eye. In contrast, mice injected with IFNλ activated LCN‐2‐/‐ neutrophils showed fewer cells infiltrating the eye. LCN‐2 promotes inflammation and AMD‐like pathology by interacting with Disabled homolog2 (Dab2) and modulating integrin β1 levels to stimulate adhesion and transmigration of activated IL‐28R1 (IFNλ receptor) positive neutrophils into the retina. Inhibiting AKT2‐dependent signaling in the mouse model neutralizes IFNλ inflammatory signals, reducing LCN‐2‐mediated neutrophil infiltration into the retina and reversing early AMD‐like phenotype changes, thereby providing a potential therapeutic target for early AMD.

Confocal Laser Scanning Microscopy of Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos.

After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde-fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, both of which correlate to apoptotic activity. Thus, LT is a good indicator of apoptosis visualized by confocal microscopy. Results of LT staining of apoptotic cell death correlate well with other whole mount apoptosis vital dyes such as Nile blue sulfate and neutral red, with the added benefit of being fixable in situ. Nile blue sulfate can also be used as a non-vital, nonspecific dye to visualize general morphology. Stains such as acridine orange can be used for surface staining of fixed embryos to yield confocal images that are similar to scanning electron micrographs. Mouse embryos were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Embryos are mounted on depression slides, and serial sections are imaged by confocal microscopy, followed by 3-D reconstruction. Embryos or tissues as thick as 500microns (μm) can be visualized after clearing with BABB. LysoTracker staining reveals apoptotic regions in organogenesis-stage mouse embryos. Morphological observation of tissue was facilitated by combining autofluorescence with Nile blue sulfate staining of fixed embryos or opaque surface staining with acridine orangestaining. The use of BABB for clearing LT vital-stained and fixed embryos matches the refractive index of the tissue to the suspending medium, allowing increased penetration of laser light in a confocal microscope. Nile blue sulfate used as a non-vital dye provides a nonspecific staining of fixed embryos that can then be cleared with methyl salicylate for confocal observation. Sample preparation and staining procedures described here, with optimization of confocal laser scanning microscopy, allow for the detection and visualization of morphological structure and apoptosis in embryos up to 500μm thick, and stained specimens can be fixed and mounted on depression slides.

High-Resolution, Three-Dimensional Reconstruction of the Outflow Tract Demonstrates Segmental Differences in Cleared Eyes.

PurposeThe rate of conventional aqueous humor outflow is the highest nasally. We hypothesized that this is reflected in regionally different outflow structures and analyzed the entire limbus by high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM).MethodsWe perfused pig eyes by anterior chamber cannulation with eight lectin-fluorophore conjugates, followed by optical clearance with benzyl alcohol benzyl benzoate (BABB). RSCM and advanced analysis software (Imaris) were used to reconstruct a three-dimensional (3D), whole-specimen rendering of the perilimbal outflow structures. We performed morphometric analyses of the outflow tract from the level of the trabecular meshwork (TM) to the scleral vascular plexus (SVP).ResultsExcept for pigmented structures, BABB cleared the entire eye. Rhodamine-conjugated Glycine max agglutinin (soybean [SBA]) labeled the outflow tract evenly and retained fluorescence for months. RSCM produced terabyte-sized files allowing for in silico dissection of outflow tract vessels at a high resolution and in 3D. Networks of interconnected lumens were traced from the TM to downstream drainage structures. The collector channel (CC) volumes were 10 times smaller than the receiving SVP vessels, the largest of which were in the inferior limbus. Proximal CC diameters were up to four times the size of distal diameters and more elliptical at their proximal ends. The largest CCs were found in the superonasal and inferonasal quadrants where the highest outflow occurs.ConclusionRSCM of cleared eyes enabled high-resolution, volumetric analysis of the outflow tract. The proximal structures had greater diameters nasally, whereas the SVP was larger in the inferior limbus.

Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy.

The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain. We then imaged the hearts with a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. Utilizing the differential spectral properties of PAS, we could identify muscle and nuclear compartments in the heart. We were also able to visualize the differences between a 3-month-old normal mouse heart and a mouse heart that had undergone heart failure due to the expression of cardiac myosin binding protein-C (cMyBP-C) gene mutation (t/t). Using 2-D and 3-D morphometric analysis, we found that the t/t heart had anomalous ventricular shape, volume, and wall thickness, as well as a disrupted sarcomere pattern. We further validated our approach using decellularized hearts that had been cultured with 3T3 fibroblasts, which were tracked using a nuclear label. We were able to detect the 3T3 cells inside the decellularized intact heart tissue, achieving nuclear/cellular resolution in 3-D. The combination of labeling, clearing, and two-photon microscopy together with tiling eliminates laborious and time-consuming physical sectioning, alignment, and 3-D reconstruction.

An evaluation of meniscal collagenous structure using optical projection tomography

BackgroundThe collagenous structure of menisci is a complex network of circumferentially oriented fascicles and interwoven radially oriented tie-fibres. To date, examination of this micro- architecture has been limited to two-dimensional imaging techniques. The purpose of this study was to evaluate the ability of the three-dimensional imaging technique; optical projection tomography (OPT), to visualize the collagenous structure of the meniscus. If successful, this technique would be the first to visualize the macroscopic orientation of collagen fascicles in 3-D in the meniscus and could further refine load bearing mechanisms in the tissue. OPT is an imaging technique capable of imaging samples on the meso-scale (1-10 mm) at a micro-scale resolution. The technique, similar to computed tomography, takes two-dimensional images of objects from incremental angles around the object and reconstructs them using a back projection algorithm to determine three-dimensional structure.MethodsBovine meniscal samples were imaged from four locations (outer main body, femoral surface, tibial surface and inner main body) to determine the variation in collagen orientation throughout the tissue. Bovine stifles (n = 2) were obtained from a local abattoir and the menisci carefully dissected. Menisci were fixed in methanol and subsequently cut using a custom cutting jig (n = 4 samples per meniscus). Samples were then mounted in agarose, dehydrated in methanol and subsequently cleared using benzyl alcohol benzyl benzoate (BABB) and imaged using OPT.ResultsResults indicate circumferential, radial and oblique collagenous orientations at the contact surfaces and in the inner third of the main body of the meniscus. Imaging identified fascicles ranging from 80-420 μm in diameter. Transition zones where fascicles were found to have a woven or braided appearance were also identified. The outer-third of the main body was composed of fascicles oriented predominantly in the circumferential direction. Blood vessels were also visualized using this technique, as their elastin content fluoresces more brightly than collagen at the 425 nm wavelength used by the OPT scanner.ConclusionsOPT was capable of imaging the collagenous structure, as well as blood vessels in the bovine meniscus. Collagenous structure variability, including transition zones between structural regions not previously described in the meniscus, was identified using this novel technique.

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.

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 light(3) 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.

Alcian Blue Staining of the Mouse Fetal Cartilaginous Skeleton: Figure 1.

INTRODUCTIONThe vertebrate skeleton forms by endochondral and intramembranous bone formation. During endochondral bone formation, mesenchyme condensations give rise to cartilages that are eventually replaced by bone. However, there are some permanent cartilages that do not ossify, such as the cartilage of the trachea and articular cartilage of the joints, and intramembranous bone formation occurs directly without a cartilage template. This protocol is ideal for revealing the cartilaginous skeleton of mouse embryos between 12.5 and 16.5 days post-coitum (dpc). The cartilaginous skeleton is stained dark blue by alcian blue, whereas the other tissues of the embryo are made transparent by a clearing solution, benzyl alcohol/benzyl benzoate (BABB).

Alcian Blue Staining of Cartilage of Short-Tailed Fruit Bat (Carollia perspicillata): Figure 1.

INTRODUCTIONThis protocol is used to visualize the cartilaginous elements of the developing skeleton using alcian blue. It has been used with excellent results on the fruit bat Carollia perspicillata, as well as several other bat species at stages from CS 14 through CS 22. Staining has been shown to last at least 2 yr in benzyl alcohol:benzyl benzoate (BABB). However, staining has been observed to weaken during the course of months. It is therefore advisable to document results immediately.