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 ...

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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 ...

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 ...

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 ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

High-Quality Sample Preparation for Multiome 10X Genomics Analysis

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. 
Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. 
The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.
This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

Author Spotlight: Advancing Biomedical Research Through Single Cell Analysis 

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...

Preparation and In Vitro Differentiation of Mesenchymal Stem Cell-Encapsulated Hydrogels for Bone Formation

Begin by trypsinizing the human mesenchymal stem cells to create a cell suspension. Next, aliquot the cell suspension into a 1.5-milliliter tube to create 10 constructs. After centrifuging the suspension at 220g for three minutes, use a vacuum pump to remove the supernatant.Now, place the cell pellet on ice. Next, bring the thiol-modified hyaluronic acid, thiol-reactive crosslinker, polyethylene glycol diacrylate, and degassed water bottles to room temperature. Under sterile conditions, use a needle equipped syringe to add one milliliter of degassed water to the bottle containing hyaluronic acid.Vortex the solution, occasionally heating it to 37 degrees Celsius until it becomes clear. Then, place the solution on ice. Under sterile conditions, add 5 milliliters of degassed water to the crosslinker bottle.Dissolve by inverting repeatedly before placing it on ice. Now, add 120 microliters of the dissolved hyaluronic acid solution to the aliquoted cell pellet and resuspend the cell pellet by pipetting the solution back and forth. Add 30 microliters of the dissolved crosslinker solution into the tube with the resuspended cell pellet.Ensure proper mixing by tapping on the tube. After mixing, briefly spin down the solution. Drop 15 microliters of the combined solution onto a paraffin-coated 24-well plate.Allow it to solidify at 37 degrees Celsius for 30 minutes. For the in vitro differentiation, add 0.5 milliliters of chondrogenic differentiation medium to the cell seated hydrogels. After three weeks, switch to the hypertrophic differentiation medium, adding 0.5 milliliters per well.Chondrogenic differentiation resulted in sulfated glycosaminoglycans in type 2 collagen positive extracellular matrix in both the hyaluronic acid or, HA, and collagen constructs. However, the distribution was more homogenous in the hyaluronic acid constructs. The collagen constructs demonstrated peripheral cells with heterogeneous morphology.The average roundness of cells in the HA constructs was higher than in the collagen constructs. Calcium deposition was detected at the outer edges of both constructs at five weeks. In the in vivo HA constructs, all implants were attached to each other in subcutaneous pockets and formed integrated bone tissue.40%of the in vivo collagen constructs were independent. Osteoid tissue with lamellar morphology was formed in the outer regions of both in vivo constructs at eight weeks post-implantation. Loss of cartilage was also observed in the HA construct.Multiple in vivo HA constructs appeared to form integrated bone tissue as indicated by the connection to the bone tissues and the presence of joint fibrous tissue. While 40%of the collagen constructs were united, the remaining constructs existed apart or were only attached without bony tissue connections. Both constructs showed positively labeled vessels in the bone marrow, and the inner cartilage was surrounded by multinucleated osteoclast cells.The mineral was deposited in the outer osteoid region of both constructs with higher mineral volume and HA constructs due to their larger size.