Bh 2 microscope

A. ovalisporum UAM-MAO strain [35 (link)] was used. Three independent experiments were performed with batch BG11 [36 (link)] (pH 8; 20 mM HEPES) cultures, at 28 °C, with continuous white light of 60 μmol photons m-2 s-1, and constant aeration. Four different culture conditions were assayed: a control, with nitrate as nitrogen source (BG11), and three cultures supplemented with 1 mM Arg, Gly, or both. Samples were withdrawn every 24 h for nine days, and all of them analyzed in triplicate. Cell observations were done with an Olympus BH-2 microscope at 400× magnification equipped with a Leica DC300F digital system.

Whole-mount RNA in situ hybridization was carried out using probes made by in vitro transcription with T7 or SP6 RNA polymerase (Promega Corporation, Madison, WI, USA). Templates were generated by PCR using the following primers: 5′-GTCTAATCCTTGCCGTCACC-3′ (Forward, SP6), and 5′-TAAGAACTTCCGCCCAGATG-3′ (Reverse, T7), common to the v1 and v2 isoforms. PCR was performed on cDNA from 1 dpf wild-type embryos. The probe and template sequences were verified.
For histological analysis, stained embryos were fixed in 4% paraformaldehyde, dehydrated, wax embedded, sectioned (8 μm) with microtome (Leitz 1516) and stained with eosin. Images were taken with an Olympus BH2 microscope, equipped with a Leica DFC 320 digital camera and IM50 software (Leica).

Experimental offspring mice were euthanized by CO₂ inhalation. Visceral white adipose tissue was immediately collected and fixed in 10% formalin/PBS. The tissue was embedded in paraffin blocks after processing and was cut into 5-μm-thick slices using a TC-2 tissue sectioner (Sorvall Instruments). Tissue slices were mounted onto positive pre-charged glass slides to ensure optimal adhesion. CD68 (Acris-antibodies, San Diego, CA), CD206 (Abcam, Cambridge, MA) and CD11c (Abcam, Cambridge, MA) were immunostained using a VECTASTAIN Elite Avidin/Biotin-Complex (ABC) kit for mouse IgG or a VECTASTAIN Elite ABC kit for rabbit IgG (Vector Laboratory). Staining of the tissue was visualized under an Olympus BH-2 microscope and pictures were taken using a Leica M165FC camera and Leica Application Suit V3 was used for picture processing. The number of positive cells per 1000 adipocytes in each staining section was blindly counted. The average of the five counts was used for data analysis. Adipocyte diameter was measured using Image J software. Two hundred cells were randomly counted in each sample. The adipocytes of five animals from each group were counted. An average diameter was recorded for each animal.