The authors would like to acknowledge the assistance of Ms Sue Connell for her expertise in resin embedding and Ms Stephania Tombs for her laboratory expertise. The authors would like to thank Professor Frank Kandziora and Dr Marie-Anne Polboth for their advice.

<ol>
 <li>The tissue is placed in a sealed container containing 10% phosphate buffered formalin solution3. Ensure minimal exposure to light, if fluorochrome analysis is to be performed, as the fluorochromes are light sensitive. </li>
 <li>Establish which orientation of the tissue will be required. </li>
 <li>Trim the specimen to size, in the correct orientation, using a band saw. Ensure safety measures are adhered to. The specimen is then placed into an opaque container, the volume of which should be approximately ten times the size of the specimen to achieve adequate fixation. The specimen should remain in the formalin solution a total of between one and two weeks depending on its size. It is suggested using control tissue for testing and optimisation purposes.</li>
 <li>Check the tissue fits into the embedding mould and place the tissue into a sealed opaque container containing 70% ethanol. </li>
 <li>Process the tissue in ascending concentrations of ethanol, shielding from light and under constant agitation.
 <ol class="lalpha">
 <li>70 % ethanol 1 week</li>
 <li>80 % ethanol 1 week</li>
 <li>90 % ethanol 1 week</li>
 <li>100 % ethanol 1 week</li>
 </ol>
</li> 
 <li>The tissue is then cleared in butanol for 1 week, again shielding from light and under constant agitation.</li>
 <li>The density of the resin and bone should be closely matched. The resin density can be altered (see step 8) and we recommend embedding a test specimen first to ensure correct density. We use Technovit 7100 (Heraeus Kulzer) for light microscopy and fluorochrome analysis. If immunohistochemistry is required an alternative resin must be used such as Technovit 9100 (Heraeus Kulzer). </li>
 <li>The Technovit 7100 preparation solution may be mixed with small aliquots of the softening solution provided with the kit, if required, but to no more than 5% the volume of preparation solution; in our experience the softening solution is rarely required. Equal parts of absolute ethanol and base liquid Technovit 7100 are mixed and applied to the tissue as a pre-infiltration solution for 24 hours minimising light exposure and with agitation.</li>
 <li>The infiltration solution is then made up using 1 g hardener I (= 1 bag) which is dissolved in 100 ml base liquid. This replaces the pre-infiltration solution and is allowed to infiltrate for 1 week under constant agitation. </li>
 <li>Place the tissue into the mould, with the cutting surface face down. Ensure there is a margin (ideally at least 5 millimetres) around the tissue for the resin; this ensures block strength. Mix 15mls of Technovit 7100 preparation solution with 1 ml of hardener II and pour around specimen in the mould and cover it by at least 5 millimietres. Curing will commence within 2 hours but will be complete overnight.</li>
 <li>Mounting is then performed by fashioning a backing block using Technovit 3040 (Heraeus Kulzer). Mix Technovit 3040 in a volume ratio of 2 parts powder to 1 part liquid to obtain a viscous liquid. Pour Technovit 3040 into the recess at the back of the block to a level of at least 2 mm above the base of the block. After about 10 minutes the mould can be peeled away and the block is ready.</li>
 <li> The density and therefore the cutting properties of methacrylate resin are susceptible to the ambient humidity in the room. The blocks should therefore be stored in a dessicator.</li>
 <li>Ground sectioning produces larger sections between 20-50 micrometres in thickness. It is useful for fluorochrome analysis (see section below) as thicker sections produce brighter fluorescence. Secure the diamond blade to macrotome. Add lubricant, such as petroleum spirit to the reservoir on the macrotome. Secure the block in the clamp and orientate to blade. Allow grinding to occur slowly (approximately 20-30 minutes per section) ensuring adequate lubrication. The first section exposes the cutting face and orientates the section- it should be discarded.</li>
 <li>The next section is then placed on a Superfrost Plus slide. The section has a tendency to curl so we recommend placing a portion of a sandwich bag over it and then holding it flat with another slide clamped in place. It is then placed into an incubator of between 60-80 degrees Celsius for 1 hour. This softens the methacrylate and helps the section adhere to the slide in a flat manner. </li>
 <li>The ground section may be cleaned and polished as required using fine grit sandpaper in a gentle manner.</li>
 <li>The sledge microtome cuts thin sections, which provides the best sections for light microsocopy. Use a sledge microtome as it has added rigidity, which is required for cutting these hard blocks. Alternatively a powered rotary microtome can be used. Use a sharp stainless steel blade. It is preferable to have two blades available as one can be sharpened as the other is used. The blade should make a 45 degree angle with the block. Surface softening can be achieved by applying a wet paper to the block if required. It may take several sections to achieve the desired cutting conditions to obtain a quality section. The section is then floated on a water bath and placed on a slide as in 16 above.</li>
 <li>Staining reagents are listed in Materials below. Protocols are based on Bancroft4 et al. Due to thickness variation it is recommended to optimise stains on test slides. Rack staining may cause tissue to float off the slide so adding the stains directly to a flat slide may be required. An identical protocol or rack staining is necessary for consistent staining outcomes required for histomorphometry.
 <ol class="lalpha">
 <li>Haematoxylin and Eosin
 <ol class="lroman">
 <li>Stain with Haematoxyilin 5-10 minutes</li>
 <li>Wash well with Scott s tap water</li>
 <li>Stain with Eosin 5 minutes</li>
 <li>Wash in tap water</li>
 <li>Clear in xylene</li>
 <li>Mount</li>
 <li>Results 
 <ol>
 <li>Osteoid = pink</li>
 <li>Calcified bone = purplish brown</li>
 <li>Nuclei = blue</li>
 </ol></li>
 </ol></li>
 <li>Von Kossa
 <ol class="lroman">
 <li>Place in silver nitrate solution and expose to strong light until mineralised bone turns black (approx 10 mins)</li>
 <li>Wash in distilled water three times</li>
 <li>Threat with sodium thiosulfate for 5 minutes</li>
 <li>Wash in distilled water</li>
 <li>Counterstain with Safrinin O</li>
 <li>Clear in xylene</li>
 <li>Mount</li>
 <li>Results
 <ol>
 <li>Mineralised bone = black</li>
 <li>Osteoid = red/pink</li>
 </ol></li>
 </ol></li>
 <li>Masson Goldner s Trichrome
 <ol class="lroman">
 <li>Wash with alkaline alcohol (90mls of 80% ethanol and 10mls of 25% ammonia for 20 minutes)</li>
 <li>Rinse in water</li>
 <li>Rinse in distilled water</li>
 <li>Stain with Weigert s Haematoxylin for 10 minutes</li>
 <li>Rinse in distilled water</li>
 <li>Stain with Ponceau-Fuchsin final solution 5 minutes</li>
 <li>Rinse with 1% acetic acid 15 seconds</li>
 <li>Stain in phosphomolybdic acid-orange G solution 5 minutes</li>
 <li>Rinse with 1% acetic acid 15 seconds</li>
 <li>Stain with light green 5 minutes</li>
 <li>Rinse with 1% acetic acid 3 changes</li>
 <li>Rinse in distilled water</li>
 <li>Mount</li>
 <li>Results
 <ol>
 <li>Mineralised bone = green</li>
 <li>Osteoid = red/orange</li>
 <li>Nuclei = blue grey</li>
 <li>Cartilage = purple</li>
 </ol></li>
 </ol></li>
 </ol></li>
 <li>Fluorochrome preparations:</li>
 <li>These are administered by slow intravenous injection, at least two weeks apart.</li>
 <li>Doses
 <ol class="lalpha">
 <li>Calcein Green 10 mg/kg IV</li>
 <li>Oxytetracycline 50 mg/kg IV</li>
 <li>Alizarin Complexone 30 mg/kg IV</li>
 </ol></li>
 <li>Preparation of Calcein Green
 <ol class="lalpha">
 <li>One gram of Calcein Green is titrated with 1 M NaOH until dissolved. </li>
 <li>pH is adjusted with 1% NaOH until pH = 7.1-7.2.</li>
 <li>Filter and keep shielded from the light</li>
 </ol></li>
 <li>Preparation of Alizarin Complexone
 <ol class="lalpha">
 <li>Three grams of Alizarin complexone is titrated with 1 M NaOH until dissolved. </li>
 <li>pH is then adjusted with 1% HCl or NaOH until pH = 7.1-7.2.</li>
 <li>Filter and keep shielded from the light</li>
 </ol>
 </li>
 <li>Preparation of Oxytetracycline
	<ol class="lalpha">
 <li>Oxytetracycline is available as an off the shelf anti-microbial drug. No preparation is required.</li>
 </ol>
 </li>
 </ol>

<ol>
	<li>Kerr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of Functional Histology. , 164-168 (2000).</li><li>Parfitt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+histomorphometry:+standardization+of+nomenclature,+symbols,+and+units.+Report+of+the+ASBMR+Histomorphometry+Nomenclature+Committee.">Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.</a> J Bone Miner Res. 2, 595-610 (1987).</li><li>An, Y. H., Martin, K. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of histology methods for bone and cartilage. , (2003).</li><li>Bancroft, J. D., Gamble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and Practice of Histological Techniques. , 352-360 (2008).</li></ol>

Solvents:
 <ul>
 <li>10% Normal Buffered Formalin, Ethanol, Xylene and Butanol acid 
 All histology grade, sulfur free. Grale Scientific Pty Ltd. http://www.grale.com.au</li>
</ul>
Moulds:
 <ul>
 <li>Peel away, plastic moulds Grale Scientific</li>
</ul>
Slides:
 <ul>
 <li>SUPERFROST PLUS Microscope slides Lomb Scientific Pty Ltd. 
 <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a></li>
 </ul>
Resin:
 <ul>
 <li>GlycolMethacrylate Technovit 7100 Heraeus Kulzer 
 <a href="http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm">http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm</a></li>
 <li>Backing Block Resin- Technovit 3040 Histo block Heraeus Kulzer</li>
 </ul>
Stains 
(Reagents from Grale Scientific)
Goldner's Trichrome stain</li>
1. Weigert's iron hemotoxylin (DeGroat)
<table style="margin-left:40px;" border="0">
<tr><td colspan="2" style="font-weight:bold;">a) Hemotoxylin solution</td></tr>
<tr><td>Hemotoxylin</td><td>1g</td></tr>
<tr><td>Absolute Alcohol</td><td>100mL</td></tr>
<tr><td colspan="2" style="font-weight:bold;">b) Iron Solution</td></tr>
<tr><td>30% aqueous ferric chloride</td><td>4mL</td></tr>
<tr><td>Hydrochloric acid (concentrated)</td><td>1mL</td></tr>
<tr><td>distilled Water</td><td>95mL</td></tr>
</table>
Filtered and added to equal volume of hemotoxylin immediately before stain used.
2. Ponceau-fuchsin-azophloxin stock solutions
<table style="margin-left:40px;" border="0">
<tr><td colspan="2" style="font-weight:bold;">a) Ponceau de xylidine solution</td></tr>
<tr><td>Ponceau de xylidine</td><td>0.75g</td></tr>
<tr><td>acid fuchsion</td><td>0.25g</td></tr>
<tr><td>Acetic Acid</td><td>1mL</td></tr>
<tr><td colspan="2">mix, and add distilled water (100mL)</td></tr>
<tr><td colspan="2" style="font-weight:bold;">b) azophloxin solution</td></tr>
<tr><td>Azophloxin</td><td>0.5g</td></tr>
<tr><td>Acetic acid</td><td>0.6mL</td></tr>
<tr><td colspan="2">mix, and add distilled water (100mL)</td></tr>
<tr><td colspan="2" style="font-weight:bold;">c) Final working stain solution</td></tr>
<tr><td>Ponceau-fuchsin solution</td><td>5-10mL</td></tr>
<tr><td>Azophloxin</td><td>2mL</td></tr>
<tr><td>0.2% acetic acid solution</td><td>88mL</td></tr>
</table>
3. Light Green Solution
<table style="margin-left:40px;" border="0">
<tr><td>Light Green</td><td>1g</td></tr>
<tr><td>acetic acid</td><td>1mL</td></tr>
<tr><td colspan="2">Mix, and add distilled water (500mL)</td></tr>
</table>
4. Phophomolybdic acid-Orange G solution
<table style="margin-left:40px;" border="0">
<tr><td>Phosphomolybdic acid</td><td>3g</td></tr>
<tr><td>Orange G</td><td>2g</td></tr>
<tr><td colspan="2">Dissolve in 500mL of distilled water and add crystal thymol.</td></tr>
</table>
Von Kossa Stain
<table style="margin-left:40px;" border="0">
<tr><td>1% aqueous silver nitrate</td></tr>
<tr><td>2.5% sodium thiosulfate</td></tr>
<tr><td>1% safranin O</td></tr>
</table>
Alcian blue stain
<table style="margin-left:40px;" border="0">
<tr><td>Alcian blue 8GX</td><td>1g</td></tr>
<tr><td>3% acetic acid solution</td><td>100mL</td></tr>
</table>
Hemotoxylin and Eosin stain
 <table style="margin-left:40px;" border="0">
<tr><td colspan="2" style="font-weight:bold;">a)Harris's hemotoxylin</td></tr>
<tr><td>Hemotoxylin</td><td>2.5g</td></tr>
<tr><td>Absolute alcohol</td><td>25mL</td></tr>
<tr><td>Potassium Alum</td><td>50g</td></tr>
<tr><td>distilled water</td><td>500mL</td></tr>
<tr><td>mercuric oxide</td><td>1.25g OR</td></tr>
<tr><td>Sodium iodate</td><td>0.5g</td></tr>
<tr><td>Glacial acetic acid</td><td>20mL</td></tr>
<tr><td colspan="2" style="font-weight:bold;">b) Eosin solution</td></tr>
<tr><td colspan="2">Commercially produced Eosin was used.</td></tr>
</table>
All stain dye powders are Gurr dye powders. 
 All reagents are analytical reagents, and not Lab reagents
Fluorochromes
<table border="1" cellspacing="0" cellpadding="0">
 <tr>
 <td> 
 Manufacturer </td>
 <td>Fluorochrome</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Sigma-Aldrich, Sydney</td>
 <td>Calcein Green</td>
 <td>C0875</td>
 </tr>
 <tr>
 <td>Sigma-Aldrich, Sydney</td>
 <td>Alizarin Complexone </td>
 <td>A3882</td>
 </tr>
 <tr>
 <td>Intervet Australia, Victoria</td>
 <td>Oxytetracycline 
 (off the shelf antimicrobial drug)</td>
 <td>Tradename; Engemycin 100</td>
 </tr>
</table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Watch this Scientific Journal Video about Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis at JoVE.com

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

undecalcified-bone-preparation-for-histology-histomorphometry

Nanyang Technological University

Research

JoVE Journal

Biology

27.1K Views.  Monash University. Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Video: Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...