Bx50 epifluorescence microscope

Water samples were pre-filtered through a 20-μm mesh plankton net and fixed in a 2% final concentration of formaldehyde (freshly prepared by filtering through 0.2 μm syringe filter) for at least 3–4 h before filtration. 50 ml of epilimnion and 100 ml of hypolimnion samples were filtered through polycarbonate filters (pore size 0.8 μm, diameter 25 mm, Advantec), rinsed twice with 1X PBS and twice with MilliQ water, air dried and frozen at −20°C until further processing.
CARD-FISH was performed according to the method described in Mukherjee et al. (2015) (link). The filters were embedded in 0.1% low-gelling-point agarose and cut into eight sections, which were hybridized at 35°C for 12 h with a 0.5 μg ml^–1 probe concentration and a 30% concentration of formamide. The probes (Supplementary Table S1) were purchased from Thermo Electron Co. (Ulm, Germany). Counting was performed using an Olympus BX50 epifluorescence microscope under 1000 × magnification at blue/UV excitation. For the kinetoplastids, either 100 microscopic fields were counted, or when the densities were low the complete filter piece was screened per sample. The total eukaryotes were counted simultaneously with the kinetoplastid cells by DAPI staining under UV excitation.

Labile zinc was measured in coronal sections from the fetal rat brain at E19. Slides were overlaid with a solution of 25 μM zinquin in Hank’s balanced salt solution (HBSS) and incubated at 37°C for 40 min. After washing in HBSS, coverslips were mounted with a solution of 90% glycerol and 10% HBSS and imaged on an Olympus BX50 epifluorescence microscope provided with a Cool-Snap digital camera. Image pro software (Rockville, MD, USA) was used to analyze the resulting micrographs. Three randomly selected fields were measured per animal and experimental condition (n = 3).

WPMY-1 fibroblasts were plated at 50% confluency. The following day, the cells were treated with 5–40 µM EGCG or luteolin +/−5 ng/ml TGF-β for 24 hours. DiIC16 (Molecular Probes, Invitrogen) was added directly to the media at a final concentration of 1 µM and incubated at room temperature for 2 minutes. Cells were fixed with 4% paraformaldehyde, washed and nuclei stained with DAPI. Images were captured on an Olympus BX-50 epifluorescence microscope using MetaMorph software.

At least 30 metaphase spreads per individual were analyzed to confirm the 2n, karyotype structure and the FISH results. Images were captured using an Olympus BX50 epifluorescence microscope (Olympus Corporation, Ishikawa, Japan) with the CoolSNAP system software and the images were processed using Image Pro Plus 4.1 Software (Media Cybernetics, Silver Spring, MD, USA). Final images were optimized and arranged using Adobe Photoshop, version 7.0. Chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st), or acrocentric (a), according to their arm ratios (Levan et al., 1964 (link)).