HB-vSMCs were preincubated with Alexa Fluor 488-conjugated CTB (1 mg ml−1, Invitrogen, C34775) for 2 d, followed by washing three times and maintained in the fresh SMCM medium for another 2 d. Before adding the freshly dissociated cortical neurons, the SMCM medium was replaced by neural basal medium. Time-lapse imaging was performed 7 d after seeding neurons. The retrograde tracing states were recorded using a motorized fluorescence microscope (Olympus, IX83). A control experiment was performed to exclude the possibility of CTB diffusing into the supernatant neural basal medium and then binding to co-cultured neurons. The supernatant medium of the pre-stained HB-vSMCs was collected and added to primary neurons. Notably, under such experimental settings, we did not observe any CTB+ neurons.
Alexa fluor 488 conjugated ctb
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488-conjugated CTB is a fluorescently labeled cholera toxin subunit B (CTB) product. CTB is a protein that binds to GM1 ganglioside receptors on cell surfaces. The Alexa Fluor 488 dye provides a green fluorescent label for visualizing and tracking CTB binding and internalization in cells.
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5 protocols using alexa fluor 488 conjugated ctb
1
Retrograde Tracing of Neuron-Smooth Muscle Co-culture
2
Labeling Striatal Neurons and Projections in Mice
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Striata of adult mice were stereotactically injected with 2 μL lentivirus with a titer of 0.5–2 × 109 particles/mL. Injection coordinates were as follows: anterior/posterior, +0.8 mm; medial/lateral, ±2.0 mm; and dorsal/ventral from skull, −3.0 mm.
To label pre-existing neurons, 2 μL of pAAV–Syn–EGFP with a titer of 1.9 × 1012 particles/mL was injected into the bilateral striata.
To label iN cells that projected into the globus pallidus, 0.3 μL of Alexa Fluor 488-conjugated CTB (Invitrogen, C34775) was injected into the globus pallidus of iDTR tMCAO model mice at 10 wk after CD68–ND1–P2A–Cre lentivirus injection. Injection coordinates were as follows: anterior/posterior, −0.5 mm; medial/lateral, ±1.9 mm; and dorsal/ventral from skull, −4.0 mm. Five days later, the animals were fixed.
To label pre-existing neurons, 2 μL of pAAV–Syn–EGFP with a titer of 1.9 × 1012 particles/mL was injected into the bilateral striata.
To label iN cells that projected into the globus pallidus, 0.3 μL of Alexa Fluor 488-conjugated CTB (Invitrogen, C34775) was injected into the globus pallidus of iDTR tMCAO model mice at 10 wk after CD68–ND1–P2A–Cre lentivirus injection. Injection coordinates were as follows: anterior/posterior, −0.5 mm; medial/lateral, ±1.9 mm; and dorsal/ventral from skull, −4.0 mm. Five days later, the animals were fixed.
3
Fluorescent Labeling of Sperm
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Following incubation and fixation, samples were labeled with 2 μg/ml of Alexa Fluor 488‐conjugated CTB (Thermo Fisher, Waltham, MA; catalogue # C34775). After 10 min, 5 μl of the labeled sperm were placed on a microscope slide, overlaid with a cover slip (50 mm no. 1), and moved to an imaging station.
4
Sperm Capacitation Induction Protocols
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Two treatments were prepared for each of 10 semen samples. Cap and Non‐Cap treatments were incubated, respectively, with and without 2‐hydroxypropyl‐β‐cyclodextrin for 3 hr. For seven of these samples, a third treatment, Cap + ionophore, was prepared in which sperm were incubated with 2‐hydroxypropyl‐β‐cyclodextrin for 2.75 hr, then the calcium ionophore A23187 (Sigma–Aldrich, Allentown, PA; reference C7522) was added to a final concentration of 20 μM and the cells were incubated for another 0.25 hr.
In a second set of experiments, Cap (n = 4) and Non‐Cap (n = 5) treatments were incubated, respectively, with and without 2‐hydroxypropyl‐β‐cyclodextrin for 3 hr, plus a third treatment of Non‐Cap + ionophore was prepared. For this treatment, sperm were incubated in basal non‐capacitating media for 2.75 hr, then the calcium ionophore A23187 (Sigma–Aldrich) was added to a final concentration of 20 μM and the cells were incubated for another 0.25 hr.
Following incubation, the sperm were attached to slides for 0.25 hr, labeled for 10 min with 10 μg/ml of Alexa Fluor® 647‐conjugated PNA from Arachis hypogaea (Thermo Fisher, Allentown, PA; reference L32460), washed 1× with mHTF, fixed for 0.5 hr, and then labeled with 2 μg/ml of Alexa Fluor 488‐conjugated CTB (Thermo Fisher, reference C34775). All labeling and slide work was done in a humidified chamber maintained at 37°C.
In a second set of experiments, Cap (n = 4) and Non‐Cap (n = 5) treatments were incubated, respectively, with and without 2‐hydroxypropyl‐β‐cyclodextrin for 3 hr, plus a third treatment of Non‐Cap + ionophore was prepared. For this treatment, sperm were incubated in basal non‐capacitating media for 2.75 hr, then the calcium ionophore A23187 (Sigma–Aldrich) was added to a final concentration of 20 μM and the cells were incubated for another 0.25 hr.
Following incubation, the sperm were attached to slides for 0.25 hr, labeled for 10 min with 10 μg/ml of Alexa Fluor® 647‐conjugated PNA from Arachis hypogaea (Thermo Fisher, Allentown, PA; reference L32460), washed 1× with mHTF, fixed for 0.5 hr, and then labeled with 2 μg/ml of Alexa Fluor 488‐conjugated CTB (Thermo Fisher, reference C34775). All labeling and slide work was done in a humidified chamber maintained at 37°C.
5
Retrograde Labeling of Phrenic Motor Neurons
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