Cell-attached and whole-cell patch clamp recordings were performed on mice under 1 – 1.5% isoflurane anesthesia on a 37ºC heating pad (DC Temperature Control System, FHC). Patch pipettes with resistance values between 5 – 7.5 MΩ were prepared by pulling filamented borosilicate glass capillaries (Warner or WPI) using a micropipette puller (Flaming-Brown P97 model, Sutter Instruments or PC-10 vertical puller, Narishige). These pipettes were filled with an internal solution containing (in mM): 135 K-gluconate, 4 KCl, 10 HEPES, 10 Na2-phosphocreatine, 4 MgATP, 0.3 Na3GTP (pH adjusted to 7.3 – 7.4 with KOH; osmolarity 280 – 290 mOsm), and 50 μM Alexa 488 dye (ThermoFisher; for pipette visualization under the two-photon microscope) or 125 K-Methanesulfonate, 7 KCl, 10 HEPES, 2 MgATP, 2 Na2ATP, 0.5 Na2GTP, 0.05 EGTA (pH adjusted to 7.3 with KOH; osmolarity 280–290 mOsm), and 50 μM Alexa 488 dye. Fully manual patch clamp experiments (Figures S4A–S4C ) were performed following previously reported protocols (Häusser and Margrie, 2014 (link); Komai et al., 2006 (link)).
Alexa 488 dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 488 dye is a fluorescent dye commonly used in fluorescence-based applications. It has an excitation maximum of 495 nm and an emission maximum of 519 nm, making it suitable for detection using standard green fluorescent protein (GFP) filter sets. The dye is widely used in a variety of research applications, including cell labeling, immunoassays, and flow cytometry.
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Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (6 mentions)
Slide warmer, by Thermo Fisher Scientific (3 mentions)
Fluoromount g, by Southern Biotech (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Maleimide reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Alexa594 dye, by Thermo Fisher Scientific (2 mentions)
Pc 10 vertical puller, by Narishige (1 mentions)
Hep 2 slides, by Bio-Rad (1 mentions)
Dapi counterstain, by Thermo Fisher Scientific (1 mentions)
Rhodamine conjugated α bungarotoxin, by Merck Group (1 mentions)
Nis element 4, by Nikon (1 mentions)
Cryostat, by Leica (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Ab146642, by Abcam (1 mentions)
Axoporator 800a, by Molecular Devices (1 mentions)
Carrageenan, by Merck Group (1 mentions)
Vibratome, by Leica (1 mentions)
Collagenase, by Merck Group (1 mentions)
Ab53841, by Abcam (1 mentions)
26 protocols using alexa 488 dye
1
Patch Clamp Recordings in Anesthetized Mice
2
Immunofluorescence Staining of Cell Markers
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The MCF‐7 and MDA‐MB‐231 cells were added to a four‐chamber slide glass and incubated overnight. The cells were then washed with phosphate‐buffered saline (PBS) and fixed with ice‐chilled methanol for 30 minutes and permeabilized with 0.2% Triton X‐100 in PBS for 30 minutes. After washing twice with PBS, the permeabilized cells were placed in 5% normal horse serum in PBS for 30 minutes (to block the nonspecific adsorption of antibodies) before incubating with anti‐CLDN1 (1:50), anti‐CLDN4 (1:50), anti‐BHLHE41 (1:50) and anti‐Myc (1:50) antibody at 4°C overnight. The cells were then incubated for 1 hour with Alexa 488 dye or Alexa 488 dye (Molecular Probes, Inc) conjugated to goat anti‐rabbit IgG antibody, while nuclear staining was performed using Hoechst 33258. The cells were observed using an Olympus IX51 fluorescent microscope (Olympus), and the images were captured with a COOLPIX 5400 camera (Nikon).
3
Tracing Neuronal Projections in Mice
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Mouse pups (P3 or P9) were anesthetized by placing them in a glove and briefly immersing them in crushed ice and water for 5-7 min. Adult mice (P29) were anesthesized with Avertin (0.025 mL/g). During the surgery, the hypothermic pups and anesthesized mice were placed on an ice pack (3-4 °C). Pups and mice received intravitreal injections of cholera toxin-β subunit (CTβ) conjugated to Alexa 488 dye (ThermoFisher Scientific, Cat no. C22841, 0.5% in Dulbecco’s phosphate buffered saline) into the left eye and cholera toxin-β subunit (CTβ) conjugated to Alexa 555 dye (ThermoFisher Scientific, Cat no. C22843, 0.5% in Dulbecco’s phosphate buffered saline) into the right eye (0.5 μL per eye for P3 pups, 1 μL for P9 pups and P29 mice). After the surgery, pups were placed in a slide warmer (Thermo Fisher scientific, Waltham, Massachusetts) at 33°C for at least 1 hour. After 24 hours, pups were transcardially perfused with PBS, then with 4% paraformaldehyde (PFA). Brains were postfixed in 4% PFA for overnight, cryoprotected in 30% sucrose, and then sectioned coronally at 40 μm, mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, Massachusetts), and coverslipped with Fluoromount-G (SouthernBiotech, Birmingham, AL).
4
Anti-nuclear Immunofluorescence of Lyn-/- Mice
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Anti-nuclear immunofluorescence was performed as previously described(23 (link)). Briefly, Kallestad HEp-2 slides (BIO-RAD) were stained overnight with 1µL of serum from either Lyn−/−IgD+/− or wildtype mice. After washing, antibody binding was detected using an anti-mouse secondary antibody conjugated to Alexa488 dye (Thermo) and DAPI counterstain (Thermo). Cells were imaged with the Crest LFOV Spinning Disk/ C2 Confocal at 1000x magnification under identical camera exposure and laser settings for knockout and control mice. Micrograph exposure was normalized to secondary only negative control in FIJI and applied to all images at once.
5
Tracing Neuronal Projections in Mice
6
Fluorescent Labeling of EC12 Peptide
7
Intracellular Alexa488 Dye Injection
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For the intracellular injection of Alexa488 dye (Thermo Scientific), we fixed slice cultures with 4% paraformaldehyde for 30–40 min. Micropipettes filled with Alexa488 solution (5–10 mM) were inserted into the cell bodies or thick primary dendrites of CA1 pyramidal neurons, after which a current (0.3–0.4 μA) was applied to the neurons for 3–5 min to deliver the fluorescent dye. The tip resistance of the micropipettes was 100–300 MΩ. The dye-loaded neurons were subsequently imaged, and the spine density was measured for the neurons treated with AP-V and CNQX (Fig. 2 ).
8
Neuromuscular Junction Visualization
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Twenty-micrometre sections were obtained by cutting isopentane fresh-frozen tibialis anterior muscle perpendicular to the muscle axis with a cryostat at −20 °C (Leica, Nanterre, France). Acetylcholine receptors in the postsynaptic apparatus of neuromuscular junctions were labelled with rhodamine-conjugated α-bungarotoxin (Sigma–Aldrich). Gangliosides distribution was labelled with the cholera toxin sub-unit beta coupled with an Alexa488 dye (1/200, ThermoFisher). Photomicrographs were taken with a Nikon microscope and analysed with NIS Element 4.0 (Nikon).
9
Fluorescent Labeling of EC12 Peptide
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Alexa-488 dye (Thermo Fisher Scientific) was stably conjugated to the thiol groups of cysteine of the purified EC1236C/44W with a maleimide reagent (Life Technologies) according to the manufacturer’s instructions. This reaction was carried out at 4°C with mixing of Alexa-488 and EC1236C/44W peptide at an equimolar ratio overnight in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl and 1 mM tris (2-carboxyethyl) phosphine. Both dithiothreitol and β-mercaptoethanol should not be used in the reaction because each of them contains free thiols. Labeled protein was separated from free dye molecules by size exclusion chromatography (Superdex 200). The fluorescently labeled EC1236C/44W (EC12 probe or probe) was aliquoted and frozen in −80°C for in-solution and competitive binding assays.
10
Histological Analysis of Skin Grafts
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