Oocytes were labeled on ice for 45 min with 10 μM Alexa 488 C5 maleimide (Molecular Probes, Eugene, OR) in high K+ solution (in mM: 98 KCl, 1.8 CaCl2, and 5 HEPES, pH 7.6). Then the cells were washed with ND96 solution and kept on ice until recording. A CA-1B amplifier was used to record whole-oocyte currents in ND96 solution. Fluorescent signals were recorded simultaneously using a Pin20A photodiode (OSI Optoelectronics, CA), filtered using a FITC filter cube (Leica, Germany, for Alexa 488), and then amplified using a patch-clamp amplifier (EPC10, HEKA, Germany).
Fitc filter cube
Manufactured by Leica
Sourced in Germany
The FITC filter cube is a specialized optical filter set designed for fluorescence microscopy. It consists of an excitation filter, a dichroic mirror, and an emission filter. The core function of the FITC filter cube is to selectively pass light wavelengths that are suitable for the excitation and detection of fluorescein isothiocyanate (FITC) labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using fitc filter cube
1
Fluorescent Labeling of Oocytes
2
Voltage-Clamp Fluorometry in Oocytes
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For voltage-clamp fluorometry, oocytes were labeled on ice for 45 min in 10 μM Alexa 488 C5 maleimide (Life Technologies) in high-potassium depolarizing solution (in mM: 98 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes, pH 7.6). The cells were washed with ND96 and kept on ice until recording. Fluorescent signals were recorded simultaneously with the whole-cell currents in ND96 solution, using a DLMFS (Leica) upright microscope through a FITC filter cube (Leica). Light from a standard 100-W halogen bulb was focused onto the animal pole of the oocyte and emission from the cube was focused on a P20A photodiode. The current from the photodiode was amplified using an EPC10 patch amplified (HEKA), low-pass filtered at 200 Hz, and sampled at 1 kHz using Patchmaster (HEKA).
All recordings were made in room temperature (20–22°C).
All recordings were made in room temperature (20–22°C).
3
Fluorescent Labeling and Electrophysiological Recordings of Oocytes
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Oocytes were labeled with 10 μM Alexa 488 C5-maleimide or Alexa 546
C5-maleimide (Molecular Probes, Eugene, OR) in high K+ solution
(98 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.6) for 45 min on ice. After labeling,
the cells were washed with ND96 and kept on ice until recording. Recordings were
performed in ND96 solution. Fluorescence emission from the sample was focused onto
a Pin20A photodiode (OSI Optoelectronics), amplified by an EPC10 (HEKA) patch
amplifier, analog filtered at 200 Hz, sampled at 1 KHz, and recorded
simultaneously with whole oocyte currents. A FITC filter cube (Leica, Germany) was
used for Alexa 488 labeled cells and a rhodamine cube (Leica) was used for cells
labeled with Alexa 546.
C5-maleimide (Molecular Probes, Eugene, OR) in high K+ solution
(98 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.6) for 45 min on ice. After labeling,
the cells were washed with ND96 and kept on ice until recording. Recordings were
performed in ND96 solution. Fluorescence emission from the sample was focused onto
a Pin20A photodiode (OSI Optoelectronics), amplified by an EPC10 (HEKA) patch
amplifier, analog filtered at 200 Hz, sampled at 1 KHz, and recorded
simultaneously with whole oocyte currents. A FITC filter cube (Leica, Germany) was
used for Alexa 488 labeled cells and a rhodamine cube (Leica) was used for cells
labeled with Alexa 546.
4
Fluorescence-Labeling of Oocytes for Electrophysiology
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All cRNA amounts were doubled for VCF experiments in order to get higher expression level. Oocytes were incubated for 30 min on ice in 10 μM Alexa 488 C5-maleimide (Molecular Probes, Eugene, OR) in high K+ solution in mM (98 KCl, 1.8 CaCl2, 5 HEPES, pH 7.6) for labeling. Cells were washed three times with ND96 solution to remove the labeling solution, and recordings were performed in ND96 solution on the CA-1B amplifier setup. Excitation and emission lights were filtered by a FITC filter cube (Leica, Germany, for Alexa 488) and the fluorescence signals were collected by a Pin20A photodiode (OSI Optoelectronics). The signals were then amplified by an EPC10 (HEKA, analog filtered at 200 Hz, sampled at 1 kHz) patch clamp amplifier and controlled by the CA-1B amplifier to make sure fluorescence signals were recorded simultaneously with currents. All other chemicals were from Sigma-Aldrich.
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