Fixation and permeabilization steps have been described in detail previously (Collins and Haigh, 2017 (link)). Briefly, 3D cultures were fixed in 4% (v/v) paraformaldehyde for 1 h. Permeabilization and limited lipid clearing were done using 1 % [w/v] SDS/0.1 M boric acid buffer for 15 m, followed by dilution to half concentration for a further 30 m. Cultures were blocked with 10 % [v/v] foetal bovine serum (FBS), 1 % [w/v] bovine serum albumin (BSA, Sigma Aldrich) in PBS for 1 h at room temperature, and NF-L primary antibody (1 in 50 dilution; Invitrogen) and anti-mouse Alexa Fluor 647 (1 in 250 dilution; Thermo Fisher Scientific), were incubated in 1 % [v/v] FBS, 1 % [w/v] BSA in PBS overnight at 4°C (in the dark for the Alexa Fluor 647). Images were captured on a Nikon A1 fluorescence microscope.
A1 fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Nikon A1 is a high-performance fluorescence microscope designed for advanced imaging applications. It features a confocal scanning system that provides high-resolution, high-contrast images of fluorescently labeled samples. The microscope is equipped with a range of laser excitation sources and sensitive detectors to capture detailed information from a variety of fluorescent probes.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Alexa fluor 488 conjugated anti mouse igg h l, by Cell Signaling Technology (1 mentions)
Alexa fluor 555 conjugated anti rabbit igg h l, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Evos fl auto, by Thermo Fisher Scientific (1 mentions)
Dmil led inverted microscope, by Leica (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
7 protocols using a1 fluorescence microscope
1
3D Culture Immunolabeling Protocol
2
Fluorescence Microscopy-Based Neuronal Analysis
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Images were obtained using a Nikon A1 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 20× objective for axon length in cultured mouse cortical neurons and a Nikon A1 equipped with a 10× objective for cortical migration and IUE. To evaluate axon length in cultured primary cortical neurons, GFP positive cells were randomly selected from each condition, and the total axon length was traced using the Fiji plugin for ImageJ software (http://imagej.net/Fiji/Downloads ). At least three independent experiments were performed, and approximately 70 neurons were analyzed. For IUE, GFP positive cells were counted using ImageJ software (https://imagej.nih.gov/ij/download.html ). The ratios of GFP+ cells in the upper cortical plate (UpCP), lower cortical plate (LoCP), intermediate zone (IZ), subventricular zone (SVZ), and ventricular zone (VZ) of the cortex were calculated and compared between groups. All values are presented as means ± standard deviation (SD). P < 0.05 was considered significant.
3
Immunofluorescence Staining of Infected Cells
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Cell samples were harvested at indicated time points after infection and fixed with 4% paraformaldehyde. After infiltration with 0.25% Triton X-100 and blocking with 5% bovine serum albumin, the cells were incubated with indicated primary antibodies at 4℃ for 12 h and stained with Alexa Fluor 488-conjugated anti-mouse IgG (H+L) and/or Alexa Fluor 555- conjugated anti-rabbit IgG (H+L) (Cell Signaling Technology) at room temperature for 1 h. The nuclei were stained with DAPI (Sigma-Aldrich). The cells were washed five times with PBST (5 min/wash), then observed and photographed on a Nikon A1 fluorescence microscope (Nikon, Tokyo, Japan).
4
Immunofluorescent Staining and Analysis
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Immunofluorescent staining was carried out following washing in PBS and fixing in 4% (v/v) paraformaldehyde. Cells were permeabilized for 10 minutes in 0.01% (v/v) triton-X-100, then blocked with 10% (v/v) FBS and 0.1% (w/v) bovine serum albumin (BSA) in PBS for 30 minutes before being transferred in to primary antibody in a 1% (v/v) FBS and 0.1% (w/v) BSA in PBS solution overnight at 4 °C. The primary antibodies used were NF-L (Invitrogen), GFAP (Abcam) and OSP (Abcam) at a 1 in 50 dilution. Cells were washed 3× in PBS before addition of AlexFluor-488 or −647 secondary antibodies at a 1 in 250 dilution and incubated for 2 hours at room temperature in the dark. Cells were washed again 3× in PBS then mounted in Fluoromount with DAPI and allowed to cure for 24 hours before imaging. Images were collected using a Nikon A1 fluorescence microscope. For analysis of cell contact, microglia were considered ‘MNLC associated’ if they were directly contacting a neighboring cell, cells that were spatially isolated in the well were classified as ‘unassociated’.
5
Brightfield and Fluorescence Microscopy
6
Mitochondrial ROS Imaging in Cells
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MitoSOX™ (Invitrogen) stock solutions were prepared by dissolving probe in anhydrous DMSO to a final concentration of 5 mM. Working solutions were prepared in normal medium to a final concentration of 5 µM and cells were labelled for 10 minutes before imaging in fresh medium. Live cell images were collected using a Nikon A1 fluorescence microscope.
7
Cholera Toxin B Labeling of Microglia
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AlexaFluor-647 cholera toxin B (CTx-B) conjugate (Invitrogen) was diluted to 1 mg/mL in PBS for immediate use. Cells were labelled with a final concentration of 3 µg/mL in normal media for 5 minutes at 37 °C in the dark. Fluorescent images of live cells were captured immediately using a Nikon A1 fluorescence microscope. AlexaFluor-647 cholera toxin B conjugate intensity was calculated using microglial GFP to draw an outline around each individual cell then AlexaFluor-647 pixel intensity per cell was measured. The same measurement protocol was used for the sites of direct cell-cell interaction with the cell body excluded from the analysis. Between 4 and 10 fields of 10–30 cells were counted per independent repeat.
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