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

This method can help answer key questions in the field of environmental toxicology about sub-organ distributions and concentration of heavy metals within biological systems. The main advantage of this technique is that AMG is an easy to use and inexpensive methodology, that is a valuable adjuvant for investigating heavy metals in tissues. We use the tissues from dolphins and whales to demonstrate the protocol.

However, we believe the protocol could be used in a variety of animal species. Demonstrating the procedure with Wen-Ta Li will be Shun Theng, a clinician from our institute. Begin by collecting pair-matched liver and kidney tissues for AMG analysis from a stranded cetacean, and fixing the tissue in 10 times the volume of 10%neutral buffered formalin for 24 to 48 hours.

Next, use disposable stainless steel microtome blades to trim the formalin-fixed liver and kidney tissues into two-centimeter by one-centimeter, three-millimeter sections, and place both liver and kidney sections from the same individual into the same labeled cassettes. Dehydrate the sections in a tissue processor through a series of graded ethanol and xylene immersions, followed by one-hour and two-hour paraffin immersions. After the two-hour paraffin immersion, transfer the tissues to the bottoms of steel histology molds and embed the dehydrated tissue samples with fresh melted paraffin.

Chill the formalin-fixed, paraffin-embedded tissue blocks on a cold plate, until the paraffin solidifies, and trim each block at the microtome until the tissue surface is exposed. Chill the samples at minus 20 degrees Celsius for 10 minutes, and obtain five-micrometer tissue sections on the microtome. Using tweezers and brushes, lift the ribbons of tissue section as they are acquired, and float them on the surface of a 45 degree Celsius double-distilled water bath, until they can be separated, and place onto individual microscope slides.

Then, place the slides on a slide warmer for one hour, at 60 degrees Celsius. Then, place the slides in slide racks, and de-paraffinize the sections in three different staining dishes containing 200 to 250 milliliters of pure non-xylene for eight-five-and three-minute incubations. Hydrate the de-parrafinized samples in different staining dishes of graded ethanol solutions, followed by rinsing in double-distilled water.

Rinse the sections one time in PBS supplemented with 5%Triton X 100, several times in PBS alone, and one time in fresh double-distilled water for 30 seconds per wash. After the last wash, label each sample with 300 microliters of mixed silver enhancement kit solution, for no more than 15 minutes in the dark at room temperature. At the end of the incubation, wash the slides with double-distilled water, and counter-stain the sections in 300 microliters of hematoxylin per slide, for 10 seconds.

Wash the slides with running tap water. Dry them and mount the sections with mounting medium. Then, capture 10 random histological images of each tissue section, under the 40X objective of a light microscope.

To analyze the autometallography, or AMG, positivity of the samples, open the images in an appropriate image analysis software program, and select image, type, and RGB stack, to split the first image into the red, blue, and green color channels. In the blue channel, select image, adjust, and threshold, to measure the percentage of the area with AMG-positive signals in each image, manually adjusting the cut-off value for the threshold of each image based on the presence of false-positive areas in the nuclei, or red blood cells. Click analyze, and set measurements, and check the area fraction, to specify that the area fraction is recorded.

Click analyze, and measure, to display the positive percent area of each histological image percent area column of the result window. To evaluate the correlation between the results of inductively-coupled plasma mass spectroscopy and AMG-positive values, open the appropriate graphing software. Create a new project file, and select XY and correlation.

Input the results of the inductively-coupled plasma mass spectroscopy and AMG analyses, and select analysis, and correlation, to analyze the strength of association between the results of the analysis by Pearson correlation analysis. In the parameters, non-linear regression window, select a different regression model on the fit page, and select the comparison methods on the compare page. Include the extra sum of squares F test, and Akaike’s Information Criterion.

According to the results of the comparison methods, select a relatively appropriate regression model in the cetacean histological silver assay, and use the assay to estimate the silver concentrations of the cetacean liver and kidney tissues, with unknown silver concentrations. AMG-positive signals include variably-sized, brown to black granules in the cytoplasm of hepatocytes and Kupffer cells, and occasional amorphous, golden yellow to brown AMG-positive signals in the lumen and basement membrane of some proximal renal tubules. There is a positive correlation between the results of inductively-coupled plasma mass spectroscopy and AMG-positivity values in the analyzed liver and kidney tissues, with a preferred linear regression through the origin, according to the extra sum of squares F test, and Akaike’s Information Criterion.

While attempting this procedure, it’s important to remember that the AMG method is an adjuvant method, and must be used with other specific method, such as ICPMS, for monitoring the actual composition of heavy metals in tissues.