Las af 6000

At the end of experiment, mice were intracardially perfused with saline followed by 4% paraformaldehyde. Brains were removed, postfixed overnight at 4°C and cryoprotected using 30% sucrose in phopshate‐buffered saline (PBS). Brains were cut (40 μm) on a freezing microtome at a coronal plane. Immunofluorescence labelling for arginine vasopressin (AVP) was performed to delineate SCN anatomical boundaries. Briefly, following washes in 0.1 m PBS and 0.1% Triton X‐100 in PBS, sections were blocked for 1 h in 5% normal donkey serum. Then, they were incubated with primary antibody (dilution 1:5000; AVP Rabbit; AB1565; Millipore, Burlington, MA, USA) for 2 days at 4°C. After washing, sections were incubated with Alexa Fluor 488 Donkey anti‐Rabbit IgG (dilution 1:800; A‐21 206; Invitrogen, Thermo Fisher Scientific) overnight at 4°C. Finally, sections were mounted onto gelatine coated slides and cover‐slipped using ProLong Gold Antifade Mountant (Molecular Probes, Invitrogen; Thermo Fisher Scientific). Photos were taken using a DFC365 FX camera (Leica, Wetzlar, Germany) connected to a DM2500 microscope (Leica) using LAS AF6000 software (Leica). Recording sites were estimated based on stereotaxic notes, CM‐DiI marks and AVP staining in accordance with atlas of Paxinos & Franklin (2001).

Synchronized adult worms were transferred to glass slides in 15 μl of M9 solution. Samples were permeabilized, fixed and washed with absolute ethanol, then 15 μl of a 2 ng/μl solution of 4′, 6′-diamidino-2-phenylindole hydrochloride (DAPI) diluted in M9 was added. Quantitative analysis was performed on z series of images acquired using a Leica DM6000 fluorescence microscope, Leica DC 350 FX camera under the control of Leica LAS AF 6000 software. Optical sections were collected at 0.50 μm increments.

Whole overnight inducted E. coli BL21(DE3) cells were collected and the expression of the H⁵-derived fusion proteins was analysed by an in vitro assay with the fluorescent SNAP-Vista Green™ substrate (New England Biolabs, Ipswich, MA; hereinafter BG-FL), as previously described⁵⁸ (link)^,64 . The in vivo imaging was carried out as described by Merlo et al.⁵⁸ (link). Briefly, bacterial cells expressing the H⁵-SspCA onto cell surface were washed twice in PBS 1× and resuspended in 50.0 μl of the same buffer supplemented with 5.0 μM of the BG-FL. After incubation at 37.0 °C for 30.0 min, cells were washed twice, resuspended, and again incubated for 30.0 min at 37.0 °C, to allow the external diffusion of the unreacted substrate. Images were collected using a DM6 fluorescence microscope and Hamamatsu camera under the control of Leica LAS AF 6000 software; excitation and emission wavelengths used suitably for AlexaFluor488 dye were λ_ex = 490 nm; λ_em = 525 nm, respectively.

For in vivo imaging, E. coli BL21(DE3) cells transformed with pET-22b/INPN-SspCA or pET-ASL^tag plasmids were IPTG-inducted, grown overnight at 37.0 °C and finally diluted until OD_600nm of 1.0. An amount of cells corresponding to a volume of 1.0 mL of the culture was washed twice in PBS 1× and finally resuspended in 50.0 μL of the same buffer supplemented with 5.0 μM of the BG-FL substrate. After an incubation at 37.0 °C for 30.0 min, cells were washed twice, resuspended and again incubated for 30.0 min at 37.0 °C, to allow the external diffusion of the unreacted substrate. Labelling was first verified by fluorescence gel-imaging on SDS-PAGE and then spotted on poly-L-lysine coated slides for microscopy analysis.
Images were collected using a DM6 fluorescence microscope and Hamamatsu camera under the control of Leica LAS AF 6000 software; excitation and emission wavelengths used suitably for AlexaFluor488 dye were λ_ex = 490 nm; λ_em = 525 nm, respectively.