E19 rat hippocampal neuronal cultures were maintained in vitro for at least 14 days before treatment with Cyto-012. At the end of the treatment, cells were washed with ice-cold DPBS, fixed in 3.7% paraformaldehyde for 15 minutes, and permeabilized in 0.1% Triton X-100 for 5 minutes. Cells were blocked in 10% Normal goat serum for 1 hour, washed, and incubated with primary antibody diluted in 3% NGS, 1X DPBS for 1 hour. Cells were washed with 1X DBPS and exposed to secondary antibody for 1 hour, followed by three washes with DPBS and mounting for analysis. Primary antibodies used included human monoclonal against BoNT/A LC (Clone: 5A20, kindly provided by Dr. James Marks, University of California, San Francisco) and mouse monoclonal antibody against SNAP-25 (Synaptic Systems, clone 111-011). Secondary Antibodies used were goat anti-mouse Alexa Fluor® 555, and goat anti-human Alexa Fluor® 488, both obtained from eBioscience. Image scanning was performed on a Nikon LSM 510 confocal microscope equipped with argon and HeNe lasers producing excitation lines of 488 and 568 nm. Images were analyzed using Zeiss LSM confocal microscopy software (v.4.2).
Lsm 510 confocal microscope
Manufactured by Nikon
Sourced in Germany
The Nikon LSM 510 is a confocal microscope designed for high-resolution imaging. It features a laser scanning system that allows for optical sectioning of samples, enabling the capture of detailed three-dimensional images. The LSM 510 is equipped with multiple laser excitation wavelengths and a range of detectors to accommodate a variety of fluorescent probes and samples.
Automatically generated - may contain errors
Lab products found in correlation
Ds fi2 camera, by Nikon (2 mentions)
Donkey serum anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Donkey serum anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Eclipse ci microscope, by Nikon (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Bx41 microscope, by Zeiss (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
4 protocols using lsm 510 confocal microscope
1
Immunocytochemistry of BoNT/A in Rat Hippocampal Neurons
2
Quantifying Astrocyte-Endothelial Interactions in Tumor Specimens
3
Quantifying Astrocyte-Endothelial Interactions in Tumor Specimens
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Immunofluorescence staining was performed for each tumor specimen using antibodies against CD31 (anti-mouse, 1:200) (Millipore, Billerica, MA), glial fibrillary acidic protein (GFAP) (anti-rabbit, 1:1000) (Abcam, Cambridge, UK), and aquaporin 4 (AQ4) (anti-rabbit, 1:200) (Millipore). AQ4 is a marker of astrocytic foot processes and has been previously used as a reliable measure of astrocyte-endothelial cell relationship integrity [6 (link),7 (link)]. Double immunofluorescence staining was performed for CD31 and GFAP; and CD31 and AQ4, using a protocol described previously [5 (link)]. The following secondary antibodies were used at 1:1000 dilution:donkey serum anti-mouse IgG absorbing at a wavelength of 568 nm (Life Technologies, Carlsbad, CA) and donkey serum anti-rabbit IgG absorbing at a wavelength of 488 nm (Life Technologies). Immunofluorescent images were captured at ×40 magnification using a Leica (Wetzlar, Germany) LSM 510 confocal microscope, while H and E images were obtained at ×40 magnification using a Nikon (Tokyo, Japan) Eclipse Ci microscope with a Nikon DS-Fi2 camera.
4
Immunostaining of NHEKs Protocol
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NHEKs were grown on 8-well chamber slides (Thermofisher Scientific). After indicated treatments, cells were fixed in 4% paraformaldehyde (PFA) (Thermofisher Scientific) for 10 minutes and blocked with 3% bovine serum albumin, 0.2 M Glycine (Sigma) with or without 1 mg/mL saponin (Sigma) prior to incubating with primary antibodies followed by appropriate 488- or 568-coupled secondary antibodies, or 488-streptavidin conjugate. Nuclei were counterstained with DAPI. All images were taken with an Olympus BX41 microscope (widefield), Zeiss LSM510 confocal microscope, or Nikon A1R Confocal STORM microscope as indicated.
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