Lucifer yellow dye

Mice at 6–8 months were anesthetized with 0.3 mg/g Pentobarbital (Mebunat 60 mg/ml) mixed 50:50 with 0.9% NaCl. Mice were perfused transcardially on ice bedding using 10–20 ml 0.9% NaCl followed by 25–50 ml of buffer comprising 4% paraformaldehyde and 0.125% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer (NaH₂PO₄–Na₂HPO₄, pH 7.2). The brain was post-fixed in 50 ml 4.0% PFA in phosphate buffer for 4–12 h at +4°C. Coronal sections of 180–200 μm were sectioned using a vibratome. The nuclei were visualized using DAPI (4^′,6-diamino-2-phenylindole, dihydrochloride (Invitrogen). Pyramidal neurons in the motor cortex and the hippocampus were located according to the Atlas of C57BL/6 mouse brains (Hof et al., 2000 ). Selected neurons were injected by iontophoresis with lucifer yellow dye (Invitrogen) using pulled borosilicate glass tubes (World Precision Instruments). The DC current source was 2–6 nA from a dual micro-iontophoresis current generator, model 260 (World Precision Instruments). After dye loading, brain slices were transferred to a slide and mounted using Shandon PermaFluor mounting medium (ThermoFisher).

Astrocyte monocultures were grown on 13 mm coverslips (VWR) for 5–7 days; on the day of injection, the cells were pre‐treated with 20 µM TAT‐GAP19 (Tocris) for 10 min prior to, and during microinjection to reduce dye uptake through Cx43‐containing hemichannels. A single cell was then microinjected with 0.5% lucifer yellow dye (Invitrogen) and 2% neurobiotin dye (Vector) in PBS using the FemtoJet microinjector system (Eppendorf) combined with FemtoTips II (Eppendorf). Injection pressure was set to 100 hPa for 0.1 s injection, constant pressure was kept at 10 hPa, and the needle was left in the injected cell for 5 min at room temperature. Cells on coverslip were then fixed in 4% PFA for 10 min and stained for further microscopic analysis. Images were analyzed in ImageJ software; injection areas were automatically thresholded and overlayed on the DAPI channel, after which the number of nuclei within the injection area were counted.
List of antibodies/dyes for microinjected area staining (all used at 1:500 final dilution):

Anti‐lucifer yellow rabbit – Invitrogen A‐5750

Cy3‐Streptavidin – Vector SA‐1300‐1

Antibodies against Cx32 and Cx26 were purchased from Sigma (St. Louis, MO, USA) and Invitrogen (Camarillo, CA, USA), respectively. Cy3-conjugated AffiniPure goat anti-rabbit IgG antibody came from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). Lucifer yellow dye was obtained from Invitrogen (Eugene, OR, USA). Gadolinium chloride hexahydrate and other reagents were from Sigma (St. Louis, MO, USA).

The cells were cultured with normal culture medium on glass coverslips until 80%–90% confluence was reached. Then, the glass coverslips were transferred to a bath solution containing 140 mM NaCl, 1 mM MgCl₂, 5.4 mM KCl, 1.2 mM CaCl₂, and 10 mM HEPES (pH 7.2). The cells were impaled with a micropipette filled with a patch pipette solution and 4% neurobiotin dye (Mr = 322.8, charge  = +1; Vector Laboratories, Burlingame, CA, USA) or 5% lucifer yellow dye (Mr = 457, charge  = −2; Invitrogen). The patch pipette solution contained 140 mM KCl, 1 mM MgCl₂, 5 mM NaCl, and 10 mMHEPES (pH 7.4). The dye solution was microinjected for 3 min with a Picospritzer (model PLI-188; Nikon, Tokyo, Japan).
After neurobiotin dye injection, the cells were fixed, permeabilized, and stained with Cy3-streptavidin conjugate (Sigma) for tracer detection. However, after lucifer yellow dye injection, the cells required only fixation. Both types of cells were then photographed with fluorescence microscopy. The adjacent cells stained by the dye were counted to determine the extent of the intercellular tracer transfer. Three independent stable-cell clones were all used to perform the dye transfer assay. The number of coupled cells was expressed as the means ±SDs. Statistical analysis was performed with Student's t-test.

Small interference RNAs were designed using the website Block-iT RNAi Designer at https://rnaidesigner.thermofisher.com/rnaiexpress/. The sequence of this siRNA is as follows: siCELF1 (5’- GCAATGAGCGTAAACTCTT -3’). As a negative control, siRNA targeting a random sequence that does not affect the Daphnia development was used: siControl (5’- GGUUAAGCCGCCUCACAUTT-3’) [34 (link)]. The siRNA oligonucleotides were dissolved in DNase/RNase-free water (Life Technologies Inc.; Grand Island, NY, USA). Two nucleotides dTdT were added to each 3′ end of the siRNAs. The siRNAs were diluted with the injection marker 2 mM Lucifer Yellow dye (Invitrogen, Carlsbad CA, USA) to have the final concentration of 100 μM or 300 μM. The injection cocktails were injected into female or male eggs of the Dsx1-reporter strain Daphnia. Samples were then observed at 24 h after injection and collected at 48 h for RNA extraction and cDNA synthesis as previously described [30 (link)].

Integrity of the monolayer barrier was determined by measuring the passive passage of Lucifer yellow dye (Invitrogen, Thermo Fisher Scientific). The protocol described by Yamaura et al. (2016) (link), with a few modifications, was followed. Initially, Lucifer yellow was dissolved in dimethyl sulfoxide and then diluted 1,000-fold in PBS solution. The mixture was added to the apical membrane of Caco-2 cells and incubated for 1 h at 37°C. A microplate reader (Tecan) with excitation and emission of 428 and 536 nm, respectively, was used for fluorescence detection.