We thank the Taiwan Cetacean Stranding Network for sample collection and storage, including the Taiwan Cetacean Society, Taipei; the Cetacean Research Laboratory (Prof. Lien-Siang Chou), the Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei; the National Museum of Natural Science (Dr. Chiou-Ju Yao), Taichung; and the Marine Biology &#38; Cetacean Research Center, National Cheng-Kung University. We also thank the Forestry Bureau, Council of Agriculture, Executive Yuan for their permit.

The study was performed in accordance with international guidelines, and the use of cetacean tissue samples was permitted by the Council of Agriculture of Taiwan (Research Permit 104-07.1-SB-62).
1. Tissue Sample Preparation for ICP-MS Analysis
Note: The liver and kidney tissues were collected from freshly dead and moderately autolyzed stranded cetaceans24, including 6 stranded cetaceans of 4 different species, 1 Grampus griseus (Gg), 2 Kogia spp. (Ko), 2 Lagenodelphis hosei (Lh), 1 Stenella attenuata (Sa). Each stranded cetacean had a field number for individual identification. The tissue sample preparation for ICP-MS analysis followed the method established in M.H. Chen&#39;s lab, and M.H. Chen&#39;s lab conducted the ICP-MS analysis11,13,25.
<ol>
	<li>Collect liver and kidney tissues for ICP-MS analysis from stranded cetaceans and store them at &#8722;20 &#176;C until use.</li>
	<li>Collect pair-matched liver and kidney tissues from the same stranded cetaceans for AMG analysis (please see step 2).</li>
	<li>Trim the outer layer of the tissue samples collected for ICP-MS analysis with a stainless-steel scalpel. Cut the inner part of the tissue samples into small cubes (about 1 cm3) and place them in zip lock plastic bags. Normally, each bag contains 10 g of the tissues.</li>
	<li>Store the plastic bags containing tissue samples at &#8722;20 &#176;C for subsequent procedures.</li>
	<li>Put the 1 cm3 cubes samples in a freeze dry system (-50 &#176;C, Vacuum pump with a displacement of at least 98 L/min, 0.002 mBar) for at least 72 h till completely dried by weighing to the constant.</li>
	<li>Homogenize the dried cubes into powder with a homogenizer for subsequent tissue digestion.</li>
	<li>Weigh 0.3 g of homogenized freeze-dried samples in 30 mL polytetrafluoroethylene (PTFE) bottles and mix them with 10 mL of 65% w/w nitric acid.</li>
	<li>Put closures on the PTFE bottles, but leave the closures untightened. 
	Note: This allows the brown fume to form in the PTFE bottles and reflux inside the bottle for digestion until the brown fume disappears and turns clear.</li>
	<li>Heat the digested samples with a hot plate, from 30 &#176;C to 110/120 &#176;C (according to the brown fume forming condition) in the PTFE bottles for 2 to 3 weeks until the brownish gas in the PTFE bottles becomes colorless and the liquid in the PTFE bottles becomes translucent greenish pale yellow or completely clear. 
	Note: Perform the heating process in chemical fume hood.</li>
	<li>Heat the digested samples at 120 &#176;C to evaporate the nitric acid in the PTFE bottles until only 0.5-1 mL remains. 
	Note: Perform the heating process in a chemical fume hood, and always monitor the temperature increase to ensure that no brownish gas leaks from the PTFE bottles&#8217; closures.</li>
	<li>Tighten the closures and cool them at room temperature for about 1 h.</li>
	<li>Place the funnels with filter papers on 25 mL volumetric flasks and wash the remaining liquid with 1 M HNO3 to a final volume of 25 mL. 
	Note: Wash the bottle for at least three times and the closure twice.</li>
	<li>Validate the analytical quality of ICP-MS analysis by using the standard reference materials, including DOLT-2 (dog&#64257;sh liver) and DORM-2 (dog&#64257;sh muscle).</li>
	<li>Use duplicates of each analytical sample and triplicates of standard reference materials for ICP-MS analysis.</li>
	<li>Average the Ag concentrations of each analytic samples and present the data as dry weight basis concentration (&#956;g/g dry weight).</li>
</ol>
2. Tissue Sample Preparation for AMG Analysis
<ol>
	<li>Collect pair-matched liver and kidney tissues for AMG analysis from a stranded cetacean and fix them in 10% neutral buffered formalin until use. 
	Note: Store the tissue samples in plastic bottles in 10% neutral buffered formalin (NBF, pH 7.0) for 24 to 48 hours. The volume of NBF should be at least 10 times greater than the tissue volume.</li>
	<li>Trim the formalin fixed liver and kidney tissues with stainless steel disposable microtome blades and put the trimmed tissue sections in cassettes with labels. 
	Note: The size of each tissue sections should be approximately 2 cm x 1 cm and the thickness of each tissue section should not exceed 3 mm. Put the liver and kidney tissues from the same individual in the same cassette.</li>
	<li>Dehydrate the trimmed tissue sections with a tissue processor through a series of graded ethanol (70% for 1 h, 80% for 1 h, 95% for 1 h, 95% for 2 h, 100% for 1 h x 2 staining dishes, and 100% for 2 h), non-xylene (for 1 h and 2 h in different staining dishes), and immerse the dehydrated tissue samples in paraf&#64257;n (for 1 h and 2 h in different staining dishes).</li>
	<li>Place the dehydrated tissue samples in the bottoms of steel histology molds and embed the dehydrated tissue samples with paraffin.</li>
	<li>Chill the formalin fixed paraffin-embedded (FFPE) tissue blocks on cold plate until the paraffin solidifies. Trim the FFPE blocks with the microtome until the tissue surface is exposed.</li>
	<li>Chill the FFPE blocks at &#8722;20 &#176;C for 10 min. Section the FFPE blocks at 5 &#181;m by microtome.</li>
	<li>Fill a water bath with double-distilled water at 45 &#176;C. Lift the ribbons of tissue sections and make them float on the surface of the warm water by using tweezers and brushes.</li>
	<li>Separate the ribbons of tissue sections with tweezers. Place a section onto a microscope slide.</li>
	<li>Place the microscope slides on a slide warmer and allow sections to dry overnight at 37 &#176;C.</li>
	<li>Put the microscope slides in slide racks and deparaffinize them by soaking them in 3 different staining dishes of pure non-xylene (approximately 200 to 250 mL) for 8, 5, and 3 min.</li>
	<li>Hydrate the tissue sections in slide racks by soaking them in different staining dishes of graded ethanol solutions (100% ethanol twice, 90% ethanol once and 80% ethanol once [1 min each]), and rinse them in double-distilled water. 
	Note: These solutions are approximately 200 to 250 mL in different staining dishes. For each wash, 30 sec is enough.</li>
	<li>Rinse the tissue sections in phosphate-buffered saline (PBS) with 0.5% Triton X-100, wash them with PBS for several times, and then rinse them in double-distilled water. 
	Note: These solutions are approximately 200 to 250 mL in different staining dishes. For each was, 30 sec is enough.</li>
	<li>Prepare equal amounts of the three components (initiator, moderator, and activator) provided by silver enhancement kit in dark and mix them thoroughly. 
	Note: The solutions of moderator and activator are sticky, so please use pipette with wide tip openings (or cut the tips to create wider openings). For each slide, 300 &#956;L of the mixed solution (depending on the size of the tissue section) is usually enough. Therefore, if 10 slides are used, the amount of each component (initiator, moderator, and activator) is 1000 &#956;L (the mixed solution is 3000 &#956;L for 10 slides).</li>
	<li>Incubate the tissue sections in the mixed solution for 15 min in the dark at room temperature. Fully cover the tissue sections on the slides with the mixed solution. A longer incubation time may lead to false-positive AMG signals.</li>
	<li>Wash the slides with double-distilled water and stain them in hematoxylin for 10 s as a counterstain.</li>
	<li>Wash the slides with running tap water, dry them, and mount them with mounting medium.</li>
	<li>Examine the slides under a light microscope.</li>
	<li>Randomly capture ten histological images with a 40X objective lens from each tissue section by using a connected digital camera with computer imaging software.</li>
</ol>
3. Semi-Quantitative Analysis for AMG Positive Values of Histological Images
Note: AMG positive value means the percentage of the area with AMG positive signals.
<ol>
	<li>Use image analysis software (ImageJ) to analyze the histological images.</li>
	<li>Open the histological image by pressing File | Open.</li>
	<li>Split the chosen picture into three color channels (red, blue, and green) by pressing Image | Type | RGB Stack.</li>
	<li>Quantify the AMG positive signals by using the blue channel. Nuclear false positive signals are usually decreased under the blue channel when hematoxylin stain is applied for nuclear counterstain (Figure 2).</li>
	<li>Measure the percentage of the area with AMG positive signals in each histological image with the threshold tool (Image | Adjust | Threshold).</li>
	<li>Manually adjust the cut-off value of the threshold for each histological image (from 90 to 110) based on the presences of false positive areas in nuclei and/or red blood cells. 
	Note: In default setting, the AMG positive signals should be highlighted in red.</li>
	<li>Press Analyze | Set Measurements, and check the box of Area Fraction to specify that the area fraction is recorded.</li>
	<li>Press Analyze | Measure. The positive percent area of each histological image is displayed in the column of %Area of the Result window.</li>
	<li>Average the positive percent areas of 10 histological images from each tissue section and define the result as the AMG positive value for each tissue section.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58232/58232fig2.jpg" /> 
Figure 2: The presence of nuclear false positive signals under different color channels (counterstain: hematoxylin stain). Representative nuclear false positive signals are indicated by yellow arrows. PPA = positive percentage of areas. <a href="https://www.jove.com/files/ftp_upload/58232/58232fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Establishment of the Cetacean Histological Ag Assay (CHAA) by Regression Model
Note: The following analysis is performed in Prism 6.01 for Windows.
<ol>
	<li>Evaluate the correlation between the results of ICP-MS and AMG positive values.</li>
	<li>Open the software, create a new project file, and choose XY and Correlation.</li>
	<li>Input data including the results of ICP-MS and AMG positive values.</li>
	<li>Press Analysis and choose Correlation under the category XY Analysis to analyze the strength of association between the results of the ICP-MS and AMG positive values by Pearson correlation analysis. 
	Note: The results of the ICP-MS and AMG positive values have to be positively correlated with each other; otherwise, the subsequent regression model should not be developed.</li>
	<li>Statistically compare the regression models, including linear regression, quadratic regression, cubic regression, and linear regression through origin, through statistics software12,26,27. 
	Note: If the regression model generates an unrealistic Ag concentration, the regression model should be abandoned12.</li>
	<li>Go back to the Data Table (left panel) and press Analysis | Nonlinear regression (curve fit) under the category XY Analysis | OK.</li>
	<li>In the window Parameters: Nonlinear regression, choose different regression model in the page Fit and then compare different regression models in the page Compare.</li>
	<li>In the page Compare, choose the comparison methods, including the extra sum-of-squares F test and Akaike&#39;s information criterion (AIC). According to the results of the comparison methods, use a relatively appropriate regression model in the CHAA.</li>
	<li>Estimate Ag concentrations of the cetacean liver and kidney tissues with unknown Ag concentrations by using the CHAA.</li>
	<li>Evaluate the accuracy and precision of the CHAA for liver and kidney tissues. The difference between precision and accuracy is illustrated in Figure 3.</li>
	<li>Accuracy: Calculate the mean standard deviation (SD) from differences between known and estimated Ag concentrations.</li>
	<li>Precision: Perform repeated measurement (at least triplicate) of AMG positive values of serial sections from the same FFPE tissues. Calculate the mean SD of measurements from liver or kidney tissues from differences between known and estimated Ag concentrations 
	Note: The methods of evaluating the accuracy and precision are depicted in Figure 4.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58232/58232fig3.jpg" /> 
Figure 3: The difference between accuracy and precision. Accuracy means how close the measurement is to the true value (i.e., Ag concentration determined by ICP-MS); precision means the repeatability of the measurement (i.e., the consistency among the repeated measurements of AMG positive values from the triplicate tissue sections). <a href="https://www.jove.com/files/ftp_upload/58232/58232fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58232/58232fig4.jpg" /> 
Figure 4: The scheme depicting the methods of evaluating the accuracy and precision. CHAA = cetacean histological Ag assay; FFPE = Formalin-fixed, paraffin-embedded; ICP-MS = inductively coupled plasma mass spectroscopy; Ai = Each of the Ag concentrations determined by ICP-MS of each pair-matched tissue sample; Bi = Each of the Ag concentrations estimated by CHAA of each pair-matched tissue sample; Ci, Di, and Ei = Each of The Ag concentrations estimated by CHAA of triplicate samples from each pair-matched tissue sample; i = 1 to n. Please see raw data of the accuracy and precision tests in the section of representative results. <a href="https://www.jove.com/files/ftp_upload/58232/58232fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Estimation of Ag Concentrations by CHAA.
<ol>
	<li>Collect the liver and kidney tissues from stranded cetaceans and fix them in 10% neutral buffered formalin.</li>
	<li>Process the formalin-fixed tissues routinely (please see step 2).</li>
	<li>Estimate the Ag concentrations of the cetacean liver and kidney tissues with unknown Ag concentrations by CHAA (please see steps 3 and 4).</li>
</ol>

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S., Danscher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autometallographic+tracing+of+mercury+in+frog+liver.">Autometallographic tracing of mercury in frog liver.</a> Environmental Pollution. 129 (2), 299-304 (2004).</li><li>Stoltenberg, M., Larsen, A., Kemp, K., Bloch, D., Weihe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autometallographic+tracing+of+mercury+in+pilot+whale+tissues+in+the+Faroe+Islands.">Autometallographic tracing of mercury in pilot whale tissues in the Faroe Islands.</a> International Journal of Circumpolar Health. 62 (2), 182-189 (2003).</li></ol>

The authors have nothing to disclose.

The purpose of the article study is to establish an adjuvant method to evaluate the Ag distribution at suborgan levels and to estimate Ag concentrations in cetacean tissues. The current protocols include 1) Determination of Ag concentrations in cetacean tissues by ICP-MS, 2) AMG analysis of pair-matched tissue samples with known Ag concentrations, 3) Establishment of the regression model (CHAA) for estimating the Ag concentrations by AMG positive values, 4) Evaluation of the accuracy and precision of CHAA, and 5) Estimat...

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their great antimicrobial effects1,2. Therefore, the production of AgNPs and the number of AgNP-containing products are increased over time3,4. However, AgNPs may be released into the environment and accumulate in the ocean5,6. They have become the major source of Ag contamination, and the public awareness of the environmental toxicity of Ag is increasing.
The status of AgNPs and Ag in the marine environment is complicated and constantly changing. Previous studies have indicated that AgNPs can remain as particles, aggregate, dissolve, react with different chemical species, or be regenerated from Ag+ ions7,8. Several types of Ag compounds, such as AgCl, have been found in marine sediments, where they can be ingested by benthic organisms and enter the food chain9,10. According to a previous study conducted in the Chi-ku Lagoon area along the southwestern coast of Taiwan, the Ag concentrations of marine sediments are extremely low and similar to the crustal abundance, and those of fish liver tissue are usually below the detection limit (&#60; 0.025 &#956;g/g wet/wet)11. However, previous studies conducted in different countries have demonstrated relatively high Ag concentrations in the livers of cetaceans12,13. The Ag concentration in the livers of cetaceans is age-dependent, suggesting that the source of Ag in their bodies is most likely their prey12. These findings further suggest the biomagnification of Ag in animals at higher trophic levels. Cetaceans, as the apex predators in the ocean, may have suffered negative health impacts caused by Ag/Ag compounds12,13,14. Most importantly, like cetaceans, humans are mammals, and the negative health impacts caused by Ag/Ag compounds in cetaceans may also occur in humans. In other words, cetaceans could be sentinel animals for the health of marine environment and humans. Therefore, the health effects, the tissue distribution, and concentration of Ag in cetaceans are of great concern.
Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost (instrument and maintenance) and the requirements for tissue storage/preparation12,15. In addition, it is usually difficult to collect comprehensive tissue samples in all investigations of stranded cetacean cases due to logistical difficulties, a shortage of manpower, and a lack of related resources12. The frozen tissue samples for ICP-MS analysis are not easily stored because of limited refrigeration space, and frozen tissue samples may be discarded due to broken refrigeration equipment12. These aforementioned obstacles hamper investigations of contamination levels in cetacean tissues by ICP-MS analysis using frozen tissue samples. In contrast, formalin fixed tissue samples are relatively easy to collect during the necropsy of dead-stranded cetaceans. Therefore, it is necessary to develop an easy to use and inexpensive method to detect/measure the heavy metals in cetacean tissues by using formalin fixed tissue samples.
Although the suborgan distributions and concentrations of alkali and alkaline earth metals may be altered during the formalin-fixed, paraffin-embedded (FFPE) process, only lesser effects on transition metals, such as Ag, have been noted16. Hence, FFPE tissue has been considered as an ideal sample resource for metal localization and measurements16,17. Autometallography (AMG), a histochemical process, can amplify heavy metals as variably sized golden yellow to black AMG positive signals on FFPE tissue sections, and these amplified heavy metals can be visualized under light microscopy18,19,20,21. Hence, the AMG method provides information on the suborgan distributions of heavy metals. It can provide important additional information for studying the metabolic pathways of heavy metals in biological systems because ICP-MS can only measure the concentration of heavy metals at the organ level18. Furthermore, digital image analysis software, such as ImageJ, has been applied to the quantitative analysis of histological tissue sections22,23. The variably-sized golden yellow to black AMG positive signals of FFPE tissue sections can be quantified and used to estimate the concentrations of heavy metals. Although the absolute Ag concentration cannot be directly determined by the AMG method with image quantitative analysis, it can be estimated by a regression model based on the data obtained from the image quantitative analysis and ICP-MS, which is named cetacean histological Ag assay (CHAA). Considering the difficulties in measuring Ag concentrations by ICP-MS analysis in most stranded cetaceans, CHAA is a valuable adjuvant method to estimate Ag concentrations in cetacean tissues, which cannot be determined by ICP-MS analysis due to the lack of frozen tissue samples. This paper describes the protocol of a histochemical technique (AMG method) for localizing Ag at the suborgan level and an assay named CHAA to estimate the Ag concentrations in the liver and kidney tissues of cetaceans.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58232/58232fig1.jpg" /> 
Figure 1: Flowchart depicting the establishment and application of cetacean histological Ag assay (CHAA) for estimating Ag concentrations. CHAA = cetacean histological Ag assay, FFPE = Formalin-fixed, paraffin-embedded, ICP-MS = inductively coupled plasma mass spectroscopy. <a href="https://www.jove.com/files/ftp_upload/58232/58232fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>HQ Silver enhancement kit</td>
			<td>Nanoprobes</td>
			<td>#2012</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgipath Paraplast</td>
			<td>Leica Biosystems</td>
			<td>39601006</td>
			<td>Paraffin</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>4026</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Xylene</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>4328</td>
			<td></td>
		</tr>
		<tr>
			<td>Silane coated slide</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>511614</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass (25 x 50 mm)</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>24501</td>
			<td></td>
		</tr>
		<tr>
			<td>Malinol</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>20092</td>
			<td></td>
		</tr>
		<tr>
			<td>GM Haematoxylin Staining</td>
			<td>Muto Pure Chemical Co., Ltd</td>
			<td>3008-1</td>
			<td></td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin solution</td>
			<td>Chin I Pao Co., Ltd</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip (1000 &#956;L)</td>
			<td>MDBio, Inc.</td>
			<td>1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPETMAN Classic P1000</td>
			<td>Gilson, Inc.</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge Tube</td>
			<td>GeneDireX, Inc.</td>
			<td>PC115-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dogfish liver</td>
			<td>National Research Council of Canada</td>
			<td>DOLT-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Dogfish muscle</td>
			<td>National Research Council of Canada</td>
			<td>DORM-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma mass spectrometry (ICP-MS)</td>
			<td>PerkinElmer Inc.</td>
			<td>PE-SCIEX ELAN 6100 DRC</td>
			<td></td>
		</tr>
		<tr>
			<td>FreeZone 6 liter freeze dry system</td>
			<td>Labconco</td>
			<td>7752030</td>
			<td>For freeze drying</td>
		</tr>
		<tr>
			<td>BRAND&#174; SILBERBRAND volumetric flask</td>
			<td>Merck</td>
			<td>Z326283</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL standard vial, flat interior with 33 mm closure</td>
			<td>Savillex Corporation</td>
			<td>200-030-12</td>
			<td>For diagestion</td>
		</tr>
		<tr>
			<td>Nitric acid, superpur&#174;, 65.0%</td>
			<td>Merck</td>
			<td>1.00441</td>
			<td>For diagestion</td>
		</tr>
		<tr>
			<td>Hot Plate/Stirrers</td>
			<td>Corning&#174;</td>
			<td>PC-220</td>
			<td>For diagestion</td>
		</tr>
		<tr>
			<td>High Shear lab mixer</td>
			<td>Silverson</td>
			<td>SL2T</td>
			<td>For homogenization</td>
		</tr>
		<tr>
			<td>Sterile polypropylene sample jar (250mL)</td>
			<td>Thermo Scientific&#8482;</td>
			<td>6186L05</td>
			<td>For homogenization</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Nikon Corporation</td>
			<td>DS-Fi2</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Nikon Corporation</td>
			<td>ECLIPSE Ni-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon&#8482; Finesse&#8482; 325 manual microtome</td>
			<td>Thermo Scientific&#8482;</td>
			<td>A78100001H</td>
			<td></td>
		</tr>
		<tr>
			<td>Accu-Cut&#174; SRM&#8482; 200 rotary microtome</td>
			<td>Sakura</td>
			<td>1429</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blade S35</td>
			<td>FEATHER&#174;</td>
			<td>207500000</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide staining dish and cover</td>
			<td>Brain Research Laboratories</td>
			<td>#3215</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel staining rack</td>
			<td>Brain Research Laboratories</td>
			<td>#3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon embedding center</td>
			<td>Thermo Scientific&#8482;</td>
			<td>S-EC</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Citadel&#174; tissue processor</td>
			<td>Thermo Scientific&#8482;</td>
			<td>69800003</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide warmer</td>
			<td>Lab-Line Instruments</td>
			<td>26005</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Shandon Capshaw</td>
			<td>3964</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Merck</td>
			<td>1541-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 6.01 for windows</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Statistic software</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel tissue embedding mould</td>
			<td>Shenyang Roundfin Trade Co., Ltd</td>
			<td>RD-TBM003</td>
			<td>For paraffin emedding</td>
		</tr>
	</tbody>
</table>

use of autometallography to localize and semi-quantify silver in cetacean tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

Representative images of the AMG positive signals in the cetacean liver and kidney tissues are shown in Figure 5. The AMG positive signals include variably-sized brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. There is a positive correlat...

Watch this Scientific Journal Video about Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues at JoVE.com

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

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7.2K Views.  National Taiwan University. A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Video: Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Use of Principal Components for Scaling Up Topographic Models to Map Soil Redistribution and Soil Organic Carbon

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it regulates the gravity-driven soil movement induced by runoff and tillage activities. The recent application of Light Detection and Ranging (LiDAR) data holds promise for generating high spatial resolution topographic metrics that can be used to investigate soil property variability. In this study, fifteen topographic metrics derived from LiDAR data were used to investigate topographic impacts on redistribution of soil and spatial distribution of soil organic carbon (SOC). Specifically, we explored the use of topographic principal components (TPCs) for characterizing topography metrics and stepwise principal component regression (SPCR) to develop topography-based soil erosion and SOC models at site and watershed scales. Performance of SPCR models was evaluated against stepwise ordinary least square regression (SOLSR) models. Results showed that SPCR models outperformed SOLSR models in predicting soil redistribution rates and SOC density at different spatial scales. Use of TPCs removes potential collinearity between individual input variables, and dimensionality reduction by principal component analysis (PCA) diminishes the risk of overfitting the prediction models. This study proposes a new approach for modeling soil redistribution across various spatial scales. For one application, access to private lands is often limited, and the need to extrapolate findings from representative study sites to larger settings that include private lands can be important.

Landscape processes are critical components of soil formation and play important roles in determining soil properties and spatial structure in landscapes. We propose a new approach using stepwise principal component regression to predict soil redistribution and soil organic carbon across various spatial scales.

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...