Application suite las

Cells were fixed with 4% paraformaldehyde (PFA) for 20 min. The cells were treated with the blocking solution (5% BSA/PBS-T) for 1 h, and then incubated with CD-31 antibody at 4 °C for 16 h, followed by Alexa Fluor^® 546 Goat Anti-Rabbit IgG antibody. Hoechst 33342 was used to nucleus counterstain. All images were acquired using the LEICA fluorescence microscope (LEICA, DM 2500) and performed with the Leica Application Suite (LAS; LEICA, version 3.8).

Cells were added 2 μg/mL of Dil-Ac-LDL and then incubated at 37 °C for 4½ h. The cells were fixed with 4% PFA for 10 min and then washed with PBS. Hoechst 33342 was used for nuclear staining. All images were explored using the LEICA fluorescence microscope (LEICA, DM 2500) and performed with the Leica Application Suite (LAS; LEICA, version 3.8).

TMRE staining was performed according to the previous study (Cho et al., 2017 (link)). TMRE was dissolved in DMSO at a concentration of 50 µM and added into fresh bacteria culture at a final concentration of 0.1 µM before seeding the plates. Worms were synchronized by egg bleach and grown on E. coli HT115 for RNAi from the hatch and transferred to RNAi plates containing TMRE at the L3/L4 stage. Worms were imaged after growing overnight on TMRE plates. TMRE staining was quantified with ImageJ. CCCP was dissolved in DMSO at a concentration of 10 mM and added into bacteria culture at a final concentration of 50 µM before seeding the plates. Images were acquired with Leica Application Suite (LAS) using Fluorescent Stereo Microscope Leica M205, with objective lens, PlanApo 5.0× LWD (10447243; Leica), and camera, Leica DFC3000 G, at room temperature.

We obtained consecutive (serial) cuts of 3-μm in the paraffined tissue block for the immunohistochemical analysis. The antigens were retrieved after deparaffinisation and rehydration in ethanol solutions. Afterwards, we blocked endogenous peroxidase activity with 0.1% H2O2 for 5 min at room temperature. The slides were incubated separately with primary antibodies against CD1a (Dako, Carpinteria, CA, USA), CD83, CD123, Factor XIIIa, CD163 (Leica Biosystems, Wetzlar, Germany), CD207 (Monosan, Uden, The Netherlands), CD209, CD68 (DakoCytomation, Glostrup, Denmark), and CD208 (Dendritics, Lyon, France) followed by the LSAB method (Dako, Carpinteria, CA, USA) and the reactions were developed by incubating the sections with 0.6 mg/ml 3,3’diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Saint Louis, MO, USA). The slides were counterstained with Carazzi’s hematoxylin. Negative controls were obtained by omitting primary antibodies. The results were observed under an optical microscope with 200× magnification (Leica Biosystems, Wetzlar, Germany). Ten consecutive and representative fields of the intraepithelial and subepithelial areas were selected for photomicrography (Leica Application Suite–LAS). Two independent examiners counted positive cells using the Image J software (version 1.52, NIH, Bethesda, Rockville, MD, USA).

After incubation with the osteoclasts, dentin slices were incubated with 10% NH₃ and sonicated for 20 cycles 30 s on/off on a Bioruptor Next-gen (Diagenode, Seraing, Belgium) at “high” intensity. After washing, the slices were incubated for 10 min with 10% KAl(SO₄)₂ and stained with PhastGel Blue R-350 coomassie tablets (GE Healthcare, Eindhoven, The Netherlands) according to the manufacturer’s protocol. For the quantification, five pictures at 200× magnification were taken from every dentin slice using the Labovert FS (Leitz, Leica, Rijswijk, The Netherlands). The Leica application Suite (LAS) was used to analyse the average bone resorption percentage per dentin slice.

Fixed tumours were rinsed with 1× PBS followed by a concessive immersing process of 15% and 30% sucrose at 4 °C overnight, respectively. Tumours were then embedded with embedding resin and frozen in cryostat section machine. Then 10-µm sections were blocked in 10% serum solution(s) at room temperature for 1 h followed by incubation with E2-Crimson (Clonetech, 632496, 1/1000, Takara Bio USA, Inc., Mountain View, CA, USA) and PECAM-1 (BD Pharmingen, 550274, 1/1000, Becton, Dickinson and Company, Oxford, UK) primary antibodies at 4 °C overnight. They were rinsed with 1× PBS and then incubated with Alexa Fluor^®-labelled secondary antibody solutions (Thermo Fisher Scientific, 1/1000) at room temperature for 2 h. The remaining protocols followed those for stemness markers staining in 4.14. Data were acquired using a Leica DM2500 fluorescence microscope and the Leica Application Suite (LAS, Leica) integrated software.

Copris lunaris males possess a long recurved cephalic horn, a carina-like anteromedial prominence that extends from the center of the pronotum, and two symmetrical lateral prominences on the pronotum [39 ]. We focused on the cephalic horn and on the carina extending from the center of the pronotum, hereafter called, for the sake of simplicity, the pronotal horn.
Cephalic horn length in Copris species has traditionally been quantified on the lateral view of the head as a straight line between the tip and base of the horn [2 (link),18 (link),20 (link)]. For a better representation of the horn curvature, we instead traced a curved line from the base to the tip of the horn (Figure 1a). This method was formerly applied with good results in other dung beetle species [19 (link)]. Pronotal horn length (Figure 1a) was measured as the length of the anterior declivity from the anterior margin of the pronotum to the dorsal tip of the pronotal carina, in accordance with former analyses [20 (link)]. All measures were captured by the LAS Measurement Module of the software Leica Application Suite (LAS), and were expressed in mm.