Axioplan light microscope

Microorganisms were isolated from two replicates per site (1–3) and substrate (bs, wb, sw). For this purpose, small amounts of sediment (ca. 0.1 cm³) and water (800 μL), respectively, were plated onto yeast malt agar plates (YMA; 1 % malt extract, 0.4 % yeast extract, 0.4 % glucose, and 1.2 % agar). Table salt (Bad Reichenhaller Markensalz, Südsalz, Germany), originating from the sampled salt-mine, was added to the medium at concentrations 4, 15, and 25 % (w/v). Media without salt addition (“0 % salt”) only contained trace amounts of salt, as included in the other ingredients. The agar plates were incubated at 15 °C and in darkness for 1 yr and examined weekly for fungal growth. Emerging mycelia were immediately transferred to fresh correspondingly composed media. Pure cultures of the strains were maintained at 4 °C and at salt concentrations where colonies exhibited maximal growth.
Cultures of P. salinarum were examined morphologically with a Zeiss Axioplan light microscope. Mycelia growing at 15 % or 25 % salinity were examined in saline water. Strains were deposited in the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSM 27530 – ex-holotype culture; DSM 27526, DSM 27527, DSM 27528, DSM 27529, DSM 27531, DSM 27532, and DSM 27533, isotype cultures) and at the CBS-KNAW Fungal Biodiversity Centre, Utrecht (CBS 138583– isotype culture).

Images of DAB- and Duplex-labeled ganglia were captured using the standard brightfield optics of a Zeiss Axioplan light microscope attached to a digital camera. Images of FastRed-labeled ganglia were acquired using the 63x oil objective of the Leica Sp5 confocal laser scanning microscope (UT Southwestern Live Imaging Core Facility). Estimates were performed using ImageJ. Specifically, we manually counted YFP-positive cells expressing select receptors and expressed the data as percentage of Nav1.8 coexpression. This was done in 2–4 left ganglia from different mice. For each gene, we counted a total of 262–536 cell profiles. Data were expressed as the percentage of YFP-positive cell profiles expressing red dots. In Duplex-labeled ganglia, we counted the number of neuronal profiles unequivocally containing green and/or red dots in 2–3 left ganglia from different mice. For each combination of gene, we counted a total of 215–572 cell profiles. Data were included in pie charts representing the percentage of cell profiles expressing green, red, or both green and red dots. Lastly, Adobe Photoshop CS5 was used to annotate, crop and adjust the contrast of our digital images.

Liver tissues from rats were fixed in 10% buffered formalin. After an overnight wash, specimens were dehydrated in graded ethanol, cleared in xylene, and paraffin-embedded. Sections 5–6 μm in thickness were obtained according to routine procedures, mounted on silane-coated slides, and stored at room temperature. Slides were dewaxed in xylene, hydrated using graded ethanol, and stained for routine histological evaluation by hematoxylin and eosin. The sections were observed with a Zeiss Axioplan light microscope (Zeiss, Berlin, Germany) and photographed with a digital camera (Canon, Hong Kong, Japan). In addition, liver sections were evaluated for apoptotic cells. Cells that showed morphological features of apoptosis (cell shrinkage, chromatin margination, and apoptotic bodies) were counted in 15 high power fields per se ction analyses.[17 (link)]

Retinal whole mounts were imaged with a Zeiss Axioplan light microscope equipped with a Zeiss Axiocam color camera using a Plan Neofluar 1.25×/0.035 objective to measure retinal surface prior and postconfocal microscopy. Image acquisition for morphometric observations in whole mounts and vertical sections was performed with a Leica TCS-SL confocal microscope equipped with 488 and 453 lasers, using a Plan Apochromat 40×/1.40 oil objective, employing identical laser and acquisition settings for all samples. For cell counts in retinal whole mounts, four fields (250 × 250 μm) were acquired at each eccentricity of the retina, altogether representing 1/15 of the total area of the tissue. Images were saved as TIFF files and transferred to a MetaMorph image analyzer, where semi-automatic cell counts were performed. The total number of cells/retina was calculated.