Axiolab

To demonstrate the apoptotic cell death in the adrenal medulla, the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay, which detects 3' hydroxyl ends in fragmented DNA as an early event in apoptotic cascade, was used. After dewaxation and rehydration of the paraffin-embedded tissue sections, stainings were performed according to the manufacturer’s instructions regarding the TUNEL assay by using the in situ cell death detection kit (Roche Applied Science, Mannheim, Germany). DNase I recombinant in the sections was used as the positive control. Label solution without terminal transferase was used in place of TUNEL reaction mixture for negative control. The positive immunostainings of TUNEL were examined by light microscope (Zeiss Axiolab, Carl Zeiss Inc., Oberkochen, Germany).

Samples were collected from each direction of the paiche (A. gigas) leather tanned with vegetable tannin and embedded in paraffin to perform the cuts for histological analysis. The samples were cut approximately 5.0 μm thick and stained using the hematoxylin-eosin (HE) technique [19 ] to describe the histology of the dermis. Histological sections were analyzed by light microscopy and photographed in a Calrs Zeiss/AxioLab and AxioxKop Zeiss photomicroscope.
For scanning electron microscopy, samples in different leather directions in relation to the length of the fish were taken to analyze the distribution of collagen fibers in the dermis and insertion coverslips and scale protection. Small samples were taken in different directions (longitudinal, transversal and diagonal), on the surface of the leather flower and on the face of the flesh. The samples were simply fixed with adhesive tape on the stabs for analysis in the SHIMADZU-SS550 scanning electron microscope, provided by the Central Research Support Complex (COMCAP/UEM). The samples were not metallized, as a low vacuum was used.

Isolated microalgae strains were enriched and purified from Australian environmental samples obtained in August 2014 by Thomas Brueck and coworkers under permission of the Australian government. Preparation of repeated serial dilution cultivations resulted in unialgal cultures after sampling. Maintained unialgal cultures were checked for purity by microscopy (Zeiss AxioLab, Carl Zeiss AG, Oberkochen, Germany) and in high throughput by fluorescence-activated cell sorting analysis (Biorad S3 Sorter, Bio-Rad Laboratories, Inc., Hercules, USA).

Mouse proximal colon tissues were fixed in 10% buffered formalin for 24 h at room temperature and subsequently embedded in paraffin. Tissues were sectioned at 5-μm thickness and deparaffinized. Sections were incubated in sodium citrate buffer and cooked in a pressure cooker for 10 min for antigen retrieval. Sections were then blocked with 5% BSA in TBS for one hour, followed incubation with the primary antibody at 4 °C overnight. After washing with TBS, sections were treated with secondary antibodies for 30 min at 37 °C, and color development was performed using the Vectastain ABC kit (Vector Laboratories, Newark, CA, USA). Sections were then counterstained with hematoxylin, and dehydrated. Images were obtained using a Zeiss AxioLab upright microscope (Carl Zeiss AG, Oberkochen, Germany) and Zeiss AxioCam ICc5 (Carl Zeiss AG, Oberkochen, Germany), and Zen2 software (Carl Zeiss AG, Oberkochen, Germany).

The samples for diatom identification were treated with hot hydrogen peroxide and hydrochloric acid following standard procedures [20 (link)] and finally mounted using Naphrax (Brunel Microscopes Ltd., Chippenham, UK) on permanent slides for species identification (Zeiss Axiolab, magnification 1000×). Taxonomic identification was based on Krammer and Lange-Bertalot [21 ], Lange-Bertalot [22 ], Krammer [23 ,24 ,25 ], Lange-Bertalot et al. [26 ], and Cantonati et al. [27 ] integrated with the paper on the Achnanthidium minutissimum species complex by Potapova and Hamilton [28 (link)]. For each sample, a minimum of 400 valves were identified using a Zeiss Axiolab microscope (Gottingen, Germania), and results were expressed as relative abundances (%). Subsequently, the α-diversity and heterogeneity of the diatomic communities for each sampling site were evaluated on the basis of the Shannon index [29 ] and Evenness [30 ], respectively.
The ecological status was assessed via calculation of the Intercalibration Common Metrics Index ICMi [31 ], which is calculated as the mean of the ecological quality ratio (EQR) of two existing indices, IPS [32 ] and TI [33 ].

The terminal deoxynucleotidyl transferase-biotin dUTP-nick end labeling (TUNEL) assay was used to detect 3’ hydroxyl ends in fragmented DNA in the hippocampus. In brief, after dewaxation and rehydration, tissue sections were processed according to the manufacturer’s instructions for the TUNEL assay with the in situ cell death detection kit (Roche, USA) and then stained with fast red dye. Sections treated with DNase I recombinant were used as the positive control. Labeling solution without terminal transferase was used in place of TUNEL reaction mixture for negative control. The positive TUNEL staining cells in dentate gyrus, subfields CA1 and CA3 were counted under a high-power magnification (20X) field of light microscope (Zeiss Axiolab, Carl Zeiss Inc. Germany). At least five fields were sampled in a section and data were expressed as the number of TUNEL-positive counts.

Necropsy was performed to evaluate histological changes produced by λ-CGN induced inflammation. Briefly, fish (n=30) per treatment were collected and fixed in 10% neutral buffered formalin for 72h at room temperature. All specimens were then decalcified in 0.5 M Ethylenedinitrilo-tetraacetic acid for 48h (44 (link)). The decalcified fish were then processed for routine paraffin axial embedding, sagittally sectioned to a thickness of 5 μm, deparaffinized following standard procedures, and stained with hematoxylin and eosin (45 (link)). This procedure allows visual assessment and analysis of the entire fish structure, with emphasize on the peritoneum, liver, pancreas, and muscle fibers. Bright field histological images were taken on a Zeiss Axiolab (Carl Zeiss, Germany) with CoolSNAP image capture program (Roper Sci. Photometrics). For each specimen, a minimum of five sections were analyzed and scored (data not shown). The calculated coefficient of variation was <10% between individuals.