Ds fi1 camera

Brains that were sectioned at the wrong angle or damaged during immunohistochemistry were excluded from the study. The location of the AVPV was determined by the presence of the anterior commissure and third ventricle, as shown in the atlas of Paxinos and Franklin (2001) . Digital images of brain regions were acquired using an Eclipse 80i microscope with a DS -Fi1 camera (Nikon, Tokyo, Japan) under 100x magnification. Only one section per animal was used to count TH immunopositive cells. The sections were counted twice independently by two observers, and the average number was used for further statistical analysis.

For each depth, 10 mL of fixed sediment trap sample (2% formaldehyde final concentration) was filtered onto black 0.2 μm pore size polycarbonate filters and allowed to dry completely. Ethanol cleansed surgical scissors were used to cut 1/8 pieces from each filter and four pieces were then positioned on a microscope slide (Fisherbrand Superfrost precleaned microscope slides #12-550-143). A 24 × 50 mm coverslip with #1.5 thickness was placed below the slide. Antifade mounting medium (Patel et al., 2007 (link)) containing 1 μg mL⁻¹ of nucleic acid stain 4′,6-diamidino-2-phenylindole (DAPI) was spotted on the coverslip (15 μL) to align with the filter pieces. The coverslip was inverted onto the slide and the filters were stained for 10 min. Filters were visualized at 1000× total magnification on a Nikon 90i epifluorescence microscope with excitation/emission settings for DAPI, chlorophyll, and phycoerythrin. Images were acquired with a QImaging Retiga EXi camera using optimized exposure times and analyzed with Nikon NIS-Elements software.
For optical microscopy, unmounted filters were visualized through a Nikon AZ100 Multizoom microscope with a 10× objective at 20× and 40× magnification (zoom settings 4 and 8) using a Nikon NI-150 illuminator. Images were captured via NIS-Elements software using a Nikon DS-Fi1 camera.

TMA slides labeled for RAB39B were subject to a semi‐automated microscope‐based capture system (Nikon Eclipse 90i microscope, DsFi1 camera and NIS elements software V 3.0, Nikon) and image analysis protocol (as previously detailed (34)). By means of a motorized software controlled stage, each tissue punch was assessed via multiple images captured at x100 magnification, forming a 3 x 3 image grid, with 15% overlap between adjacent fields such that the total area sampled for each punch was 1.7 mm². Following the capture of all 40 punches, image grids were inspected and regions of interests (ROIs) applied to exclude overt tissue abnormalities (folds and tears), tissue edges or erroneously included white matter regions. Any punch which was too damaged or missing from the array was noted and factored into the analysis. Within the established ROIs, a single restriction threshold based on RGB values was applied to capture immunoreactivity generating a binary signal image from which percentage area coverage could be calculated. For areas represented by more than one tissue punch, the resulting measures of area coverage were pooled and the mean for each region calculated.

HaCaT cells (immortalized human keratinocyte line) (CLS cell lines service, Germany, Cat nº 300493) were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (Gibco) and 50 µg/mL of gentamicin (Gibco) at 37 °C and 5% CO₂. HaCaT monolayers on a 24-well plate were scratched and imaged on a TS100 Eclipse inverted microscope (Nikon) equipped with a DS-1QM camera (Nikon). After that, HaCaT cells were treated with the peptide at a final concentration of 5 µM. Images were taken at 24, 48, and 72 h, and the area of opening was measured using ImageJ software. For each photograph, 10 measures of the open wound were recorded, averaged, and compared with the control samples. GraphPad Prism software was used for statistical analysis, performed by a Tukey test.
Groups of 20 wild-type (WT) zebrafish larvae (three days post-fertilization) were anesthetized with MS-222, submitted to a tail fin cut, and exposed to the peptide at a final concentration of 5 µM. All zebrafish larvae were imaged using a Nikon AZ100 lens equipped with a DS-Fi1 camera (Nikon). Measures of the tail fins at day 0, 2, 4, and 7 were performed, using the posterior notochord as a reference to the end of the tail.

The dissected internae were fixed with Bouin solution. Paraffin sections (5 μm thick) were made using standard histological methods on the Leica RM-2265 microtome and stained with hematoxylin-eosin. The sections were examined under a Leica DM2500 microscope, photos were taken with a Nikon DS-Fi1 camera and processed with ImageJ software (FiJi).
Interna of Polyascus polygenea (fam. Polyascidae) isolated from the host was rinsed in fresh water and incubated in silver nitrate solution for 24 hours, after that whole specimen was mount on slides. This method is used to visualize the nervous system.