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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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) Estimation of Ag concentrations by CHAA.
In this study, the data of ICP-MS were significantly and positively correlated with those of AMG positive values, suggesting that the Ag concentration in cetacean tissues can be estimated by the AMG positive value. Therefore, the CHAA, which is based on the AMG positive value and regression model, has been developed for estimating the Ag concentrations in the liver and kidney tissues of cetaceans. Generally, a regression model with more parameters (i.e., a more complex regression model) fits well into the data, but it is undetermined that the more complex one is actually better than the simpler one. Therefore, the best regression model must be chosen by statistical analysis26,27. The results of the statistical analysis indicate that the linear regression model is sufficient to estimate the Ag concentration based on the AMG positive value12.
In CHAA for kidney tissue, the mean SD (0.35) of the precision test was larger than that of the accuracy test (0.16). Conversely, in CHAA for liver tissue, the mean SD (2.8) of the precision test was smaller than that of the accuracy test (3.24). Based on this result, it is suggested that the uneven distribution of the AMG positive signals and the relatively low Ag concentrations in cetacean kidney tissue interfere negatively with the precision of CHAA for kidney tissue. Therefore, the CHAA for kidney tissue may be accurate but imprecise. However, the even distribution of the AMG positive signals and the relatively high Ag concentrations in cetacean liver tissues suggest that the CHAA for liver tissue is a reliable method to estimate the Ag concentrations in cetacean liver tissues. Furthermore, if more tissues with known Ag concentrations determined by ICP-MS are available, a more accurate and precise regression model can be developed to estimate the Ag concentration.
Although the current protocols provide an adjuvant method to investigate Ag in animal tissues, some limitations on the AMG method should be noted. First, false-positive AMG signals may present due to interference from other heavy metals, such as mercury, bismuth and zinc28. Therefore, the results of the AMG method have to be interpreted with other specific methods, such as ICP-MS, to monitor the actual composition of heavy metals28. Second, it is difficult to detect a homogenously distributed heavy metal because it may generate brighter amorphous AMG positive signals, which may not be identified by visualization under microscopic examination. Furthermore, the amorphous and brighter AMG positive signals are difficult to analyze with image analysis software because the color of the AMG positive signals may be similar to that of the background (e.g., the amorphous AMG positive signals found in the lumen of proximal renal tubules). Therefore, the AMG positive signals cannot be highlighted after the adjustment of the cut-off value of the threshold in the image analysis software. Third, because the AMG positive values are based on the percentage of the area of AMG positive signals, it is possible that the values of highly concentrated heavy metals may be underestimated.
FFPE samples are relatively easy to collect and store, and our previous study has demonstrated that the current AMG method can successfully amplify FFPE samples stored for over 15 years12. The mechanism of AMG is not affected by different animal species, for it has been wildly used in various animal species20,29,30,31. Although the current article is focused on the cetaceans, the protocols described here may also be used in different animal species. In addition, the cost of the AMG method with ICP-MS is relatively low (as compared to laser ablation-ICP-MS), and thus the current protocols are valuable for researchers or countries lacking sufficient research funding to investigate the distribution and concentration of heavy metals in animal tissues. In conclusion, the use of AMG with quantitative analysis to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

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.remotexs.ntu.edu.sg/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 &amp;mu;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&amp;reg; 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&amp;reg;, 65.0%</td><td>Merck</td><td>1.00441</td><td>For diagestion</td></tr><tr><td>Hot Plate/Stirrers</td><td>Corning&amp;reg;</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&amp;trade;</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&amp;trade; Finesse&amp;trade; 325 manual microtome</td><td>Thermo Scientific&amp;trade;</td><td>A78100001H</td><td></td></tr><tr><td>Accu-Cut&amp;reg; SRM&amp;trade; 200 rotary microtome</td><td>Sakura</td><td>1429</td><td></td></tr><tr><td>Microtome blade S35</td><td>FEATHER&amp;reg;</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&amp;trade;</td><td>S-EC</td><td></td></tr><tr><td>Shandon Citadel&amp;reg; tissue processor</td><td>Thermo Scientific&amp;trade;</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 correlation between the results of ICP-MS and AMG positivity values in liver and kidney tissues, and linear regression through origin is preferred according to the extra sum-of-squares F test and AIC12,26,27. In the accuracy test, the mean SDs of the CHAA for liver and kidney are 3.24 and 0.16, respectively. In the precision test, the mean SDs of the CHAA for liver and kidney are 2.8 and 0.35, respectively. The raw data of the accuracy and precision tests are summarized in Table 1. The AMG positive values, Ag concentrations estimated by CHAA, and Ag concentrations measured by ICP-MS from the liver and kidney tissues of these six stranded cetaceans are summarized in Table 2.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58232/58232fig5.jpg" /> 
Figure 5: Representative histological images of the AMG positive signals in the liver and kidney tissues of cetaceans (counterstain: hematoxylin stain). (A) The AMG positive signals in cetacean liver tissue are evenly distributed (Grampus griseus (Gg); &#64257;eld code: TP20111116; Ag concentration measured byinductively coupled plasma mass spectroscopy (ICP-MS): 21.82 &#956;g/g dry weight). (B) The AMG positive signals are brown to black granules of various sizesin the cytoplasm of hepatocytes (red arrows) and Kupffer cells (red arrow heads) (Gg; &#64257;eld code: TP20111116). (C) A few AMG positive signals of brown to black granules are shown in the cytoplasm of hepatocytes (red arrows) (Kogia spp. (Ko); &#64257;eld code: TC20110722; Ag concentration measured by ICP-MS: 3.86 &#956;g/g dry weight). (D) The AMG positive signals in cetacean kidney tissue are mainly located in the renal cortex (Gg; &#64257;eld code: TP20111116; Ag concentration measured by ICP-MS: 0.42 &#956;g/g dry weight). The black dashed line is placed on the junction between the renal cortex and medulla. (E) Higher magni&#64257;cation of Figure 5D (red dashed rectangle). The AMG positive signals in the renal cortex are brown to black granules of various sizes in the cytoplasm of the proximal renal tubular epithelium (red arrows). Amorphous golden yellow to brown AMG positive signals are shown in the lumens (red arrow head) and basement membrane (yellow arrow head) of some proximal renal tubules. No to minimal AMG positive signals are shown in the glomeruli (green arrow) and distal renal tubules (green arrow head)(Gg; &#64257;eld code: TP20111116). (F) Scattered brown granules of various sizes are shown in thecytoplasm of the proximal renal tubular epithelium (red arrows) (Ko; &#64257;eld code: TC20110722;Ag concentration measured by ICP-MS: 0.05 &#956;g/g dry weight). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58232/58232fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="7">Accuracy test</td>
		</tr>
		<tr>
			<td rowspan="2">Field number&#160;</td>
			<td colspan="3">Liver</td>
			<td colspan="3">Kidney</td>
		</tr>
		<tr>
			<td>CHAA*</td>
			<td>ICP-MS</td>
			<td>SD</td>
			<td>CHAA*</td>
			<td>ICP-MS</td>
			<td>SD</td>
		</tr>
		<tr>
			<td>TP20111116</td>
			<td>16.82</td>
			<td>21.82</td>
			<td>4.99</td>
			<td>0.64</td>
			<td>0.42</td>
			<td>0.22</td>
		</tr>
		<tr>
			<td>TC20110611</td>
			<td>10.12</td>
			<td>2.77</td>
			<td>0.96</td>
			<td>0.11</td>
			<td>0.05</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>TC20110722</td>
			<td>2.70</td>
			<td>3.86</td>
			<td>1.15</td>
			<td>0.01</td>
			<td>0.05</td>
			<td>0.04</td>
		</tr>
		<tr>
			<td>TD20110608</td>
			<td>0.76</td>
			<td>0.06</td>
			<td>7.35</td>
			<td>0.02</td>
			<td>0.05</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>TP20110830</td>
			<td>13.97</td>
			<td>14.93</td>
			<td>4.28</td>
			<td>0.69</td>
			<td>1.04</td>
			<td>0.24</td>
		</tr>
		<tr>
			<td>IL20110101</td>
			<td>6.00</td>
			<td>1.73</td>
			<td>0.72</td>
			<td>0.38</td>
			<td>0.14</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">Mean SD</td>
			<td>3.24</td>
			<td colspan="2">Mean SD</td>
			<td>0.16</td>
		</tr>
		<tr>
			<td colspan="7">Precison test</td>
		</tr>
		<tr>
			<td rowspan="2">Field number&#160;</td>
			<td colspan="3">Liver</td>
			<td colspan="3">Kidney</td>
		</tr>
		<tr>
			<td>CHAA*</td>
			<td>ICP-MS</td>
			<td>SD</td>
			<td>CHAA*</td>
			<td>ICP-MS</td>
			<td>SD</td>
		</tr>
		<tr>
			<td rowspan="3">TP20111116</td>
			<td>20.90</td>
			<td rowspan="3">21.82</td>
			<td rowspan="3">4.08</td>
			<td>0.21</td>
			<td rowspan="3">0.42</td>
			<td rowspan="3">0.44</td>
		</tr>
		<tr>
			<td>16.11</td>
			<td>0.22</td>
		</tr>
		<tr>
			<td>17.75</td>
			<td>0.14</td>
		</tr>
		<tr>
			<td rowspan="3">TD20110608</td>
			<td>1.52</td>
			<td rowspan="3">0.06</td>
			<td rowspan="3">1.71</td>
			<td>0.00</td>
			<td rowspan="3">0.05</td>
			<td rowspan="3">0.02</td>
		</tr>
		<tr>
			<td>2.40</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td>1.12</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td rowspan="3">TP20110830</td>
			<td>13.12</td>
			<td rowspan="3">14.93</td>
			<td rowspan="3">2.70</td>
			<td>0.45</td>
			<td rowspan="3">1.04</td>
			<td rowspan="3">0.59</td>
		</tr>
		<tr>
			<td>12.50</td>
			<td>0.26</td>
		</tr>
		<tr>
			<td>11.35</td>
			<td>0.33</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">Mean SD</td>
			<td>2.83</td>
			<td colspan="2">Mean SD</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td colspan="7">*The regression equations of the CHAA for livers and kidneys were respectively Y = 2.249 &#215; X (adjusted R2 = 0.74) and Y = 0.07288 &#215; X (adjusted R2 = 0.69).</td>
		</tr>
	</tbody>
</table>
Table 1: The representative results of the accuracy and precision tests for cetacean histological Ag assay (CHAA). CHAA = cetacean histological Ag assay, ICP-MS = inductively coupled plasma mass spectroscopy, SD = standard deviation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Field number&#160;</td>
			<td rowspan="2">Species</td>
			<td colspan="3">Liver</td>
			<td colspan="3">Kidney</td>
		</tr>
		<tr>
			<td>AMG</td>
			<td>CHAA*</td>
			<td>ICP-MS</td>
			<td>AMG</td>
			<td>CHAA*</td>
			<td>ICP-MS</td>
		</tr>
		<tr>
			<td>TP20111116</td>
			<td>Gg</td>
			<td>7.48</td>
			<td>16.82</td>
			<td>21.82</td>
			<td>8.82</td>
			<td>0.64</td>
			<td>0.42</td>
		</tr>
		<tr>
			<td>TC20110611</td>
			<td>Ko</td>
			<td>4.50</td>
			<td>10.12</td>
			<td>2.77</td>
			<td>1.52</td>
			<td>0.11</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>TC20110722</td>
			<td>Ko</td>
			<td>1.20</td>
			<td>2.70</td>
			<td>3.86</td>
			<td>0.11</td>
			<td>0.01</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>TD20110608</td>
			<td>Lh</td>
			<td>0.34</td>
			<td>0.76</td>
			<td>0.06</td>
			<td>0.21</td>
			<td>0.02</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>TP20110830</td>
			<td>Lh</td>
			<td>6.21</td>
			<td>13.97</td>
			<td>14.93</td>
			<td>9.43</td>
			<td>0.69</td>
			<td>1.04</td>
		</tr>
		<tr>
			<td>IL20110101</td>
			<td>Sa</td>
			<td>2.67</td>
			<td>6.00</td>
			<td>1.73</td>
			<td>5.26</td>
			<td>0.38</td>
			<td>0.14</td>
		</tr>
		<tr>
			<td colspan="8">*The regression equations of the CHAA for livers and kidneys were respectively Y = 2.249 &#215; X (adjusted R2 = 0.74) and Y = 0.07288 &#215; X (adjusted R2 = 0.69).</td>
		</tr>
	</tbody>
</table>
Table 2: The AMG positive values, Ag concentrations (&#956;g/g, dry weight) estimated by cetacean histological Ag assay (CHAA), and Ag concentrations (&#956;g/g, dry weight) measured by ICP-MS from the liver and kidney tissues of six stranded cetaceans. Gg = Grampus griseus, Ko = Kogia spp., Lh = Lagenodelphis hosei, Sa = Stenella attenuata.

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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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Nanyang Technological University

Research

JoVE Journal

Environment

7.2K Views.  National Taiwan University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van milieutoxicologie over suborgelverdelingen en concentratie van zware metalen binnen biologische systemen. Het belangrijkste voordeel van deze techniek is dat AMG is een eenvoudig te gebruiken en goedkope methodologie, dat is een waardevolle adjuvans voor het onderzoeken van zware metalen in weefsels. We gebruiken de weefsels van dolfijnen en walvissen om het protocol aan te tonen.Wij zijn echter van m...

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

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Developmental Biology

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Chemistry

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Bioengineering

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Medicine

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

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

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