Mice at 6 to 10 months were anesthetised 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% paraformaldehyde in phosphate buffer for 4–12 h at +4 °C. Coronal sections of 180 to 200 µm were sectioned using a vibratome. The nuclei were visualized using DAPI (4′,6-diamino-2-phenylindole, dihydrochloride; InvitrogenSelected)). 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).
Model 260
Manufactured by World Precision Instruments
The Model 260 is a precision weighing instrument designed for laboratory applications. It features a high-resolution digital display and a sturdy construction for reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Pulled borosilicate glass tubes, by World Precision Instruments (2 mentions)
Lucifer yellow dye, by Thermo Fisher Scientific (2 mentions)
Shandon permafluor mounting medium, by Thermo Fisher Scientific (2 mentions)
4 6 diamino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
S120vc sensor, by Thorlabs (1 mentions)
U 25nd25, by Olympus (1 mentions)
U 25nd6, by Olympus (1 mentions)
A6625, by Merck Group (1 mentions)
4 protocols using model 260
1
Neuronal Labeling and Visualization
2
Neuronal Morphology Visualization in Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
3
Iontophoretic Ca2+ Delivery to Endolymph
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ears with chronic endolymphatic hydrops have been shown to have elevated endolymphatic Ca2+ (Ninoyu and Meyer zum Gottesberge, 1986 (link); Meyer zum Gottesberge and Ninoyu, 1987 (link); Salt and DeMott, 1994b (link), 1997 (link); Fettiplace and Ricci, 2006 ). Administration of Ca2+ into the endolymphatic space can thus model some aspects of chronic endolymphatic hydrops. Ca2+ was iontophoresed into the endolymphatic space of the second cochlear turn using positive current. Pipettes for iontophoresis applications were made from single barreled glass with internal fiber. The pipette tip was beveled to a 2–3 μm diameter and filled with 160 mM CaCl2. The electrode tips were then filled with 0.5% agarose gel to prevent volume passive displacement of the electrolyte during the experiment (i.e., leakage of the electrolyte into the cochlea). The electrodes were stored with the tips in CaCl2 solution, allowing the electrolyte to equilibrate with the gel. Ca2+ was iontophoresed into endolymph for 15 min with 100 nA of current using a microiontophoresis current programmer (Model 260, World Precision Instruments).
4
Optogenetic Neural Stimulation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For iontophoresis of ACh, a sharp glass pipette ($15 MU) was filled with 10 mM acetylcholine chloride (A6625, Sigma) dissolved in external saline. ACh was ejected into the MB calyx by a brief (500 ms) positive current pulse using an iontophoresis unit (Model 260, World Precision Instruments).
Stimulation of optogenetic probes LED/mercury light stimulation Wide-field optical stimulation was achieved by high power LEDs (M470L2 and M590L3 for ReaChR and CsChrimson, respectively, Thorlabs) or filtered light from a mercury lamp (for Arch). An LED (M470L2) with peak output at wavelength of $470 nm was used for both the activation of ReaChR and excitation of Citrine tagged to ReaChR. Because ReaChR is sensitive to a broad spectrum of light (Lin et al., 2013) , blue light was sufficient to make the PNs expressing ReaChR fire at $200 Hz (Figure S3A). Light from an LED or a mercury lamp was collimated and delivered to an upright microscope (BX51Wl, Olympus) equipped with a 40x water-immersion objective lens (NA 0.80). LED light was pulsated at 80 Hz. Neutral-density filters (U-25ND25 or U-25ND6, Olympus) were used to stimulate the cells at lower intensities. All the reported optical intensity of LED light was measured at the back aperture of the objective lens (S120VC sensor, Thorlabs).
Stimulation of optogenetic probes LED/mercury light stimulation Wide-field optical stimulation was achieved by high power LEDs (M470L2 and M590L3 for ReaChR and CsChrimson, respectively, Thorlabs) or filtered light from a mercury lamp (for Arch). An LED (M470L2) with peak output at wavelength of $470 nm was used for both the activation of ReaChR and excitation of Citrine tagged to ReaChR. Because ReaChR is sensitive to a broad spectrum of light (Lin et al., 2013) , blue light was sufficient to make the PNs expressing ReaChR fire at $200 Hz (Figure S3A). Light from an LED or a mercury lamp was collimated and delivered to an upright microscope (BX51Wl, Olympus) equipped with a 40x water-immersion objective lens (NA 0.80). LED light was pulsated at 80 Hz. Neutral-density filters (U-25ND25 or U-25ND6, Olympus) were used to stimulate the cells at lower intensities. All the reported optical intensity of LED light was measured at the back aperture of the objective lens (S120VC sensor, Thorlabs).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!