This work was supported by a NSF RUI Grant MCB-0950372 entitled Molecular Characterization of Black Widow Spider Silks.

1. Anesthetizing the spider and isolation of the abdomen
<ol>
	<li>Transfer the spider from a glass jar into a cardboard box lined with plastic (Figure 1A). We typically collect spiders from woodpiles, garages or bushes and house them in the lab in glass jars. Because the spider can not climb up the plastic lining, it provides an efficient method to transfer the spider from a glass jar or coffee can into a smaller vial for anesthetizing purposes. While handling the spider at this point, you should be wearing two pairs of latex gloves or gardening gloves for safety purposes.</li>
	<li>While the spider is in the box lined with plastic, approach the spider from its backside and let the spider slide into the plastic culture vial. After the spider enters the vial, immediately place the plug over the top (Figure 1B). We recommend using a vial that is approximately 101.6 mm x 31.75 mm (L x D). This minimizes the amount of carbon dioxide gas needed for the anesthetizing step.</li>
	<li>Anesthetize the spider by introducing carbon dioxide gas at 5-10 pounds per square inch for 10 min (Figure 1C). This amount of gas will only knock the spider out for around 5 min, so after anesthetization you must immediately proceed to steps 1.4-1.5.</li>
	<li>Place the anesthetized spider into a small dissection dish and use forceps to position the spider dorsal side up (red hourglass facing down).</li>
	<li>Clip the pedicel (narrow stalk connecting the cephalothorax and the abdomen) to release the abdomen from the rest of the spider (Figure 1D). Store the cephalothorax in the freezer overnight in a petri dish and discard after frozen the next day. Be careful when moving the cephalothorax as the fangs of the spider are present on this segment.</li>
	<li>Fasten the abdomen to the Sylgard material in the small dissection dish using a single insect pin. Insert the insect pin through the hole made from the pedicel clipping (Figure 1E). This can easily be identified as the site where the fluid is emerging from the clipping of the pedicel. Make sure the spinneret (opposite end of the pedicel) is oriented toward the bottom (closest to you).</li>
</ol>
2. Removal of the exoskeleton
<ol>
	<li>Make the first incision through the exoskeleton using a pair of microscissors, starting at the hole from the pedicel clipping. Cut laterally until the dorsal side is reached on both sides (Figure 1F). Using a pair of forceps, peel back the anterior exoskeleton segment and insert a second pin at this location. Following the insertion of the second pin, turn the dissecting tray caudally (90&deg; angle) from the initial lateral cuts and continue cutting until the spinnerets are reached (Figure 1G). This incision will be perpendicular or vertical to the initial lateral cut. Do not cut through the spinnerets when the vertical incision is made (posterior relative to the hourglass and it has a circular appearance). Failure to insert the second pin will result in the abdomen spinning when the vertical incisions are performed.</li>
	<li>Submerge the abdomen in dissecting buffer solution (0.1 M Sodium Chloride, 0.015 M Sodium Citrate, 0.1% Diethyl Pyrocarbonate).</li>
	<li>Peel the remaining exoskeleton back starting from the initial, lateral incision. When this is completed, the fatty tissue should be clearly visible (Figure 1H).</li>
</ol>
3. Isolation of the silk-producing glands
<ol>
	<li>Using forceps begin teasing away the fat layer (Figure 1H). The fat will have a whitish to yellowish appearance. Continue until most of the fat is removed and the silk glands are clearly visible. The tubuliform gland consists of three pairs of very long, cylindrical tubes that are abundant in the abdominal cavity. The major ampullate, which is found in pairs, is a large, crescent shaped ampulla with a long convoluted distal tail and a proximal duct that decreases in width as it approaches the spider's spinnerets. The minor ampullate is very similar in morphology to the major ampullate, but it is significantly smaller. The flagelliform gland, also occurring in pairs, is round and multilobular with a small cylindrical zig-zag duct that extends down toward the spinnerets. The aggregate gland (found in pairs) is a very large, multilobular gland that has a very large excretory duct which has large, irregular lobes on it. The aciniform glands occur in large numbers and resemble short, small fingerlike projections. The pyriform gland is the smallest gland in the abdomen and has many compacted, cylindrical ducts that resemble a fan with many small excretory ducts that extend down to the spinnerets.</li>
	<li>We recommend removing the glands in the following order: tubuliform, major ampullate, flagelliform, minor ampullate, aggregate, aciniform, and then the pyriform (Figure 2A-B).</li>
	<li>Beginning with the tubuliform, tease these glands off of the rest of the glands - they should be sitting on top of the other glands (Figure 2A). There are three pairs of tubuliform glands in a single spider. Allow them to float free but remain attached to their spinnerets by their ducts.</li>
	<li>Next, tease away the major ampullate glands. There are two major ampullate glands present in one spider. During the removal procedure, make sure the ducts remain attached to the glands (Figure 2A).</li>
	<li>Continue to tease the rest of the glands away from each other, working carefully to avoid puncturing any of the glands, especially the aggregate and flagelliform glands. Each gland should be free floating all the way to its exit point in the exoskeleton to the spinnerets. These glands are also found in pairs. Remove each gland by pinching the duct with forceps and carefully pulling until it breaks away from the exit point. The major ampullate, flagelliform, minor ampullate, and aggregate glands are found in pairs. The aciniform and pyriform glands occur in many more pairs.</li>
	<li>Remove each gland by pinching the duct with forceps and carefully pulling until it breaks away from the exit point down by the spinneret. We typically store the glands wet; however, we do not add additional buffer.</li>
	<li>As the glands are removed, place them into sterile pre-labeled 1.5 ml microcentrifuge tubes. Individual glands are shown in Figure 3A-G. Keep these glands on ice and then flash freeze them in liquid nitrogen. Following flash freezing in liquid nitrogen, place them at -80&deg;C for storage.</li>
</ol>
4. Representative Results
When removing the different glands, extreme care should be taken while handling the flagelliform and aggregate glands, as these two structures can be easily punctured and damaged with the forceps. Furthermore, it is worth noting that morphologically the flagelliform and aggregate glands look very similar prior to their removal and they are often intertwined with each other. To prevent cross-contamination of these tissues upon removal, the dissector should carefully tease these structures apart. Additionally, upon the initial exposure of the silk-producing glands, the aciniform and pyriform glands will be the most difficult to immediately visualize due to their smaller size and anatomical location. Often there is the presence of many eggs. Moreover, additional care needs to be taken when removing the pyriform gland, as this tissue is extremely sticky and will often adhere to your forceps. When this procedure is done properly, it is possible to obtain all seven silk-producing glands from a single spider in a highly purified manner. Typically, one can recover microgram quantities of total RNA and protein from a single spider dissection. An example of using the total RNA for qPCR to examine the mRNA levels of a tubuliform restricted fibroin gene, TuSp1, is shown (Figure 4). Representative experiments that involve protein lysates collected from the glands e.g. in-solution tryptic digestion followed by MS analysis (could also be used for MS/MS analysis) or an amino acid composition analysis is shown (Figure 5-6, respectively).
<img src="/files/ftp_upload/2382/2382fig1a.jpg" alt="Figure 1a" /><img src="/files/ftp_upload/2382/2382fig1e.jpg" alt="Figure 1e" /> Figure 1. Handling, anesthetizing, and removal of the exoskeleton from the abdomen of a female black widow spider. A) Transfer of the spider into a cardboard box lined with plastic to facilitate moving the spider into a smaller plastic vial. B) Spider placed into a plastic vial with plug. C) Anesthetizing the spider with carbon dioxide gas. D) Separation of the cephalothorax from the abdomen using microscissors. E) Immobilization of the abdomen in the dissecting tray using insect pins. F) View after a lateral incision is made with the scissors and a portion of the exoskeleton is pealed back with forceps. G) Incisions being made perpendicular to the initial lateral cut. H) Removal of the exoskeleton and exposure of the fat layer.
<img src="/files/ftp_upload/2382/2382fig2.jpg" alt="Figure 2" /> Figure 2. Visualization of the seven different silk-producing glands while intact in the abdomen of the spider as well as surrounding tissues. A) Images of the tubuliform, major ampullate, flagelliform, aggregate, minor ampullate tissues at 12X magnification. B) Image of the aciniform and pyriform glands at 12X magnification.
<img src="/files/ftp_upload/2382/2382fig3a.jpg" alt="Figure 3a" /><img src="/files/ftp_upload/2382/2382fig3e.jpg" alt="Figure 3e" /> Figure 3. Photographs of the seven silk-producing glands after their removal from the abdomen. All images were captured at 20X magnification with the exception of the aciniform and pyriform glands, which were done at 40X. A) major ampullate gland; B) major and minor ampullate (right side) for size comparison; C) tubuliform; D) flagelliform; E) aggregate; F) aciniform; G) pyriform.
<img src="/files/ftp_upload/2382/2382fig4.jpg" alt="Figure 4" /> Figure 4. Representative results of the expression pattern of the silk gene, TuSp1, after surveying the TuSp1 mRNA levels in the different silk-producing glands (excluding the pyriform gland) using quantitative real-time PCR (qPCR) following isolation of total RNA from the glands.
<img src="/files/ftp_upload/2382/2382fig5.jpg" alt="Figure 5" /> Figure 5. Example of MS analysis of protein extracts obtained from the pyriform gland following in-solution tryptic digestion. Note: x2 represents 2-fold magnification of spectrum intensity. Spectrum peptide ion masses that correspond to regions of the PySp1 fibroin are shown with the symbol #.
<img src="/files/ftp_upload/2382/2382fig6.jpg" alt="Figure 6" /> Figure 6. Typical result of the amino acid composition profile of the proteins extracted from the tubuliform gland. Note: ASX = Asp and Asn; GLX = Glu and Gln. Blue coloration reflects amino acids with polar side chain groups whereas red coloration represents amino acid residues with non-polar side chain groups.

<ol>
	<li>Vollrath, F., Knight, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int. J. Biol. Macromol. 24, 243-243 (1999).</li><li>Gosline, J. M., Guerette, P. A., Ortlepp, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Exp. Biol. 202, 3295-3295 (1999).</li><li>Gosline, J. M., DeMont, M. E., Denny, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Endeavour. 10, 31-31 (1986).</li><li>Hinman, M. B., Jones, J. A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Trends Biotechnol. 18 (9), 374-374 (2000).</li><li>Lewis, R. V., Xu, 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> Proc. Natl. Acad. Sci. 87, 7120-7120 (1990).</li><li>Colgin, M. A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Sci. 7 (3), 667-667 (1998).</li><li>Tian, M., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Appl. Phys. A-Mater. 82, 265-265 (2006).</li><li>Hu, X., Lawrence, B., Kohler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry. 44 (30), 10020-10020 (2005).</li><li>Vasanthavada, K., Hu, X., Falick, 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=.">.</a> J Biol Chem. 282 (48), 35088-35088 (2007).</li><li>Hayashi, C. Y., Blackledge, T. A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mol. Biol. Evol.. 21 (10), 1950-1950 (2004).</li><li>Blasingame, E., Tuton-Blasingame, T., Larkin, 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> J Biol Chem. 284 (42), 29097-29097 (2009).</li></ol>

No conflicts of interest declared.

Our methodology for the microdissection of the silk-producing glands from the black widow spider offers an effective means to obtain highly purified silk-producing glands. Dissections can be completed in 1.5 to 3 hrs, yielding a complete set of the seven distinct silk-producing glands from cobweavers. Obtaining highly purified silk-gland samples allows investigators the ability to perform a broad range of biochemical studies, including identification of new silk or chaperone proteins stored within the glandular luminal...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0420-1</td>
<td>0.1 M in water</td>
</tr>
<tr>
<td>Diethyl pyrocarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-5758 - 5 ml</td>
<td>0.1% v/v</td>
</tr>
<tr>
<td>Sodium citrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S1804 - 1 kg</td>
<td>0.015 M in water</td>
</tr>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>Leica MZ16</td>
<td>&nbsp;</td>
<tr>
<td>Digital microscope camera</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>DFC320</td>
<td>Software - Leica Application Suite v2.8.1</td>
</tr>
<tr>
<td>Vannas scissors</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>500260</td>
<td>&nbsp;</td>
<tr>
<td>Stainless steel forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>501764</td>
<td>Mini Dumont #M5S</td>
</tr>
<tr>
<td>Insect pins</td>
<td>&nbsp;</td>
<td>Indigo Instruments</td>
<td>33414-2</td>
<td>Insect pins #2</td>
</tr>
<tr>
<td>Small or large dissection dishes</td>
<td>&nbsp;</td>
<td>Living Systems Instrumentation</td>
<td>DD-50-S or DD-90-S</td>
<td>52 mm diameter x 18 mm H (Sylgard Depth ~6mm) or 93 mm x 22 mm</td>
</tr>
<tr>
<td>Drosophila culture vials</td>
<td>&nbsp;</td>
<td>Carolina Biological Supply Company</td>
<td>FR-17-3076</td>
<td>Size is 31.75 mm diameter x 101.6 mm</td>
</tr>		</tbody>
		</table>
		</div>

microdissection of black widow spider silk-producing glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Watch this Scientific Journal Video about Microdissection of Black Widow Spider Silk-producing Glands at JoVE.com

Microdissection of Black Widow Spider Silk-producing Glands

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

microdissection-of-black-widow-spider-silk-producing-glands

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Biology

15.7K Views.  University of the Pacific. Здесь мы опишем эффективные стратегии для удаления шелкового желез из живота женщины черные вдовы. Эта процедура позволяет быстрое изоляции семь различных шелковых желез в высокой степени очистки моды, важный процесс для следователей изучения паук производ...

Video: Microdissection of Black Widow Spider Silk-producing Glands

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...