C plan

For cell counting, 1 μl of each water droplet was diluted in 39 μl of water (18.2 MΩ · cm water resistivity using a Milli-Q Advantage A10 device equipped with a Q-GardT2 filter, a QuantumTEX filter, and a MillipakExpress 40 0.22-μm filter; Merck Millipore, Germany). Cells were counted with a light microscope (DMLS; Leica, Germany) equipped with a 40×/0.65 NA ocular (C Plan; Leica, Germany) and with a counting chamber (Thoma; Brand GmbH + Co. KG, Germany). In total, 10 droplets from each oil seep were examined.
To validate the first counting, an additional 12 droplets from Pitch Lake were stained with 4′,6-diamidino-2-phenylindole (DAPI). To this end, 1 μl of each water droplet sampled from Pitch Lake oil was mixed with 1 ml of DAPI solution (25 μg ml⁻¹; Sigma, Steinheim, Germany), incubated for 20 min in the dark, and subsequently filtered through 0.2-μm polycarbonate membrane filters (Isopore; EMD Millipore, Cork, Ireland). Filters were stored at 4°C until further use. Cells were counted with an epifluorescence microscope (Axio scope.A1; Carl Zeiss Microscopy GmbH, Göttingen, Germany) equipped with a 100×/1.25 NA oil objective (N-Achroplan; Carl Zeiss Microscopy GmbH, Göttingen, Germany).

For the quantitative assessment of immunostaining, three nonoverlapping images were randomly captured per section using a Leica DML B2/11888111 microscope equipped with a Leica DFC450 camera and Leica C PLAN 10 × 0.22 objective or a Leica DM5500 B/11888817/12 microscope equipped with a Leica DFC450C camera and Leica HI PLAN 10 × 0.25 objective. For each image, the field of view was at 10 × magnification. The number of immunopositive cells in the fields from at least three sections/animal was counted using ImageJ software (US National Institutes of Health, Bethesda, MD, USA) and averaged per field for each animal. This was blindly performed by an independent, experienced researcher. The calculated percentages for at least eight animals per experimental group were considered for comparison and statistical analyses.