Dm500

Image acquisition was performed using the following equipment: upright light microscope Leica DM500 (Leica Microsystems, Switzerland), epifluorescence microscope Olympus MVX10 (Olympus Corporation, Japan), and the confocal microscopes Zeiss LSM 510 and Zeiss LSM 710 (Carl Zeiss AG, Germany). Image analysis and quantification were performed using the software ImageJ^{77 (link)}. In the case of IHC-samples, at least two cryosections containing the most representative areas of the injured/regenerating tissue were considered for each heart ventricle. For the analysis, regions of interest were first digitally selected to cover the entire injured/regenerating tissue starting from the amputation plane. Then, in order to analyze the fluorescens signal, fixed parameters for particle size and shape (TUNEL⁺-nuclei, mpx⁺-cells) and pre-determined signal-thresholds (TUNEL⁺-nuclei, mpx⁺-cells, mpeg1⁺-cells, fli1a⁺-vessels) were selected to allow software-assisted quantification Cardiomyocyte proliferation was determined on blinded-samples, for which descriptions of the specific morphology of the cardiomyocytes and their nuclei^{79 (link)} were used as guidelines. For normalization, the results were divided by the area of the region of interest, which considered a margin of 300 µm above the amputation plane and the regenerating tissue beyond.

The TH-labeled neurons were counted in each of the fourth section of medulla (40 of each 160 μm). All cells with cell body and at least one dendrite or axon in the VLM (A1/C1 neuronal clusters) and NTS (A2/C2neuronal clusters) were counted bilaterally to quantify the extent of anti-DβH-saporin induced lesion. The neurons (at the magnification 200x) were counted with the aid of an optical microscope (Leica DM500, Leica Microsystems, SP, Brazil) coupled to a digital image acquisition system (Leica Application Suite, V.3.10, Leica Microsystems, Brazil).

After the micro-CT measurements, the harvested samples were prepared for the histological and histomorphometric analyses. The samples were decalcified in Plank-Rychlo’s solution (MUTO Pure Chemicals Co., Tokyo, Japan) for 5 days, dehydrated in graded ethanol concentrations, and then embedded in paraffin. The embedded samples were longitudinally cut into 4-µm-thick sections and stained with hematoxylin and eosin (H&E; Sigma, St. Louis, MO, USA). For the qualitative analysis of the bone regenerative process, the stained samples were evaluated, especially in the border and center areas, using a standard light microscope (Leica DM500, Leica Microsystems, Wetzlar, Germany) connected to a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Four sites in each sample were randomly selected to calculate the new bone formation percentage by using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

Bacterial strains with nematicidal activity via fumigation were screened in a two-compartment Petri dish according to the method as described in [32 (link)] with some modifications. A 50 μL aliquot of bacterial culture (OD600 ≈ 1) was transferred onto the agar medium in one compartment, while E.coli OP50 was transferred onto the NGM agar in the other compartment. The plate was incubated at 28 ℃ for 24 or 48 h. A chunk of agar containing about 200 C. elegans was placed on the NGM agar afterward. After being sealed tightly with parafilm (Bemis, Neenah, WI, USA), the plate was incubated at 25 °C for another 24 h. The viability of the nematodes was examined under a light microscope (Leica DM500, Leica Microsystems, Germany). A three-compartment Petri dish was used to confirm the fumigant activity of bacterial VOCs by placing activated charcoals in the third compartment. The activated charcoal could adsorb volatile compounds, consequently blocking their fumigant activity.

The morphology of the isolates was observed following the conventional slide culture technique. Sterile PDA was cut into small squares of (approximately 1 cm) and a block was placed on a sterile glass slide inside Petri plates (100 × 15 mm) underlaid with sterile filter paper (Whatman 1). Each agar block was inoculated with a fungal colony on the four corners using sterile needles and a coverslip aseptically placed over it. The filter paper underlay was wetted with distilled water and incubated in the dark at 25 °C for 3 days. The coverslip was gently taken off and placed on a glass slide containing a drop of lactophenol cotton blue (LPCB) and examined under the light microscope (Leica DM500, Leica Microsystems, Wetzlar, Germany) with a 40× objective lens [39 (link)].

The purified pathogenic fungus discs (d = 6 mm) were inoculated on PDA for mycelial growth and on CLA for macroconidium production. After culturing at 25 °C for six days, the culture characteristics of hyphae and macroconidia were observed and recorded using a binocular microscope (Leica DM500, Leica Microsystems (Shanghai) Trading Co., Ltd., Shanghai, China).