mRFP-GFP-LC3B reporter THP-1 macrophages (1 × 105 cells/ml) were differentiated using PMA and infected with mycobacterial strain H37Ra (MOI of 10:1) for 6 h. After washing with PBS, the cells were cultured with or without BZA (5 μM) for 12 h. The cells were then washed twice in PBS and with 4% paraformaldehyde solution (Solarbio, China) for 15 min at room temperature. Cells were stained with Hoechst stain and mounted with antifade agent (Invitrogen, USA). The cells were visualized, and images were acquired with an Olympus FV1000 confocal microscope (Nikon A1R) and processed using Image J software (NIH, USA). A total of 30 to 40 infected cells were analyzed, and the level of autophagy was measured by enumerating the number of LC3 puncta per cell.
Fv1000 confocal microscope
Manufactured by Nikon
Sourced in United States
The FV1000 is a confocal microscope produced by Nikon. It is designed to capture high-resolution, detailed images of samples. The FV1000 utilizes a laser scanning system to excite fluorescent samples and capture the resulting emission signals.
Automatically generated - may contain errors
Lab products found in correlation
Antifade agent, by Thermo Fisher Scientific (1 mentions)
Hoechst stain, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde solution, by Solarbio (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Goat anti rabbit cy5, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Amira software, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Vt1000, by Leica (1 mentions)
Smz1500, by Leica (1 mentions)
Fn1 microscope, by Nikon (1 mentions)
Mz16f, by Leica (1 mentions)
Nis element, by Nikon (1 mentions)
Focus software, by Helicon (1 mentions)
Transmission electron microscope, by JEOL (1 mentions)
Widefield microscope, by Nikon (1 mentions)
Goat anti 5ht, by Abcam (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Anti fluorescence quenching agent, by Thermo Fisher Scientific (1 mentions)
Ab25630, by Abcam (1 mentions)
9 protocols using fv1000 confocal microscope
1
Mycobacterial Infection Modulates Autophagy
2
Quantification of IAV Infection and LC3B-II
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To allow tracking of LC3B II and viral protein, cells grown on collagen-coated coverslips were transduced with lentivirus expressing LC3B-GFP (BacMamm 2.0; catalog no. P36235, ThermoFisher), as follows.
18h post-transduction, 293T or A549 cells were infected with the indicated IAVs at an MOI of 5 PFU/cell and incubated at 37°C. At 8h or 12 h post-IAV infection, cells were washed once with PBS and fixed with 4% paraformaldehyde. Following permeabilization with 0.1% Triton X-100, cells were incubated overnight at 4°C with anti-M2 monoclonal antibody (E10; catalog no. EMS009, Kerafast). Three washes were performed prior to addition of goat anti-mouse Alexa Fluor-647 secondary antibody (catalog no. A21241, Life Technologies) and incubation for 2 h at room temperature. Antibody dilutions and washes were performed with PBS plus 1% Tween-20. Cells were treated with DAPI, and coverslips were mounted on slides using Vectashield (Vector Labs) mounting medium. Images were obtained with on a Nikon FV1000 confocal microscope at the Emory Integrated Cellular Imaging core facility.
18h post-transduction, 293T or A549 cells were infected with the indicated IAVs at an MOI of 5 PFU/cell and incubated at 37°C. At 8h or 12 h post-IAV infection, cells were washed once with PBS and fixed with 4% paraformaldehyde. Following permeabilization with 0.1% Triton X-100, cells were incubated overnight at 4°C with anti-M2 monoclonal antibody (E10; catalog no. EMS009, Kerafast). Three washes were performed prior to addition of goat anti-mouse Alexa Fluor-647 secondary antibody (catalog no. A21241, Life Technologies) and incubation for 2 h at room temperature. Antibody dilutions and washes were performed with PBS plus 1% Tween-20. Cells were treated with DAPI, and coverslips were mounted on slides using Vectashield (Vector Labs) mounting medium. Images were obtained with on a Nikon FV1000 confocal microscope at the Emory Integrated Cellular Imaging core facility.
3
Immunofluorescent Labeling of AVT in Brain
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The anti-AVT antibody was a generous gift from Dr Soojin Ryu (Johannes Gutenberg University, Mainz, Germany). Immunofluorescence labelling was carried out according to standard procedures. Brains were dissected fresh and fixed in 4% PFA for 2 days at 4 °C, and were then washed in phosphate buffered saline (PBS) and stored in methanol at −20 °C until processing. 100 μm coronal sections were collected using a vibratome (Leica VT1000 S, Leica Biosystems). After blocking in PBS containing 5% normal goat serum (Sigma, Cat. no. G9023), 1% dimethyl sulfoxide (Sigma, Cat. no. 276855) and 0.2% Triton X-100 (Fisher, Cat. no. 10254640), we incubated in primary antibody (Rabbit anti-AVT, 1:50038 (link)) for 24 h at 4 °C. The secondary antibody (Goat anti-rabbit Cy5, 1:500; Invitrogen, Cat no. A10523) was incubated for 2 h at room temperature. Brain sections were imaged at the level of the preoptic area using an Olympus FV1000 confocal microscope with a 20x Nikon objective. Images were assembled using Amira software (Thermo Scientific).
4
Microscopic Imaging of Whole Larvae
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Whole larvae images were obtained with Nikon SMZ1500 and Leica MZ16 F. Images were deconvolved with Helicon Focus software (HeliconSoft). Acquisition of confocal images stacks was performed with a Leica TCS SP2 confocal microscope equipped with 10x and 40x objectives, with an Olympus FV1000 confocal microscope equipped with 10x and 60x objectives, Nikon Ti Spinning Disc confocal microscope equipped with 20x and 40x objectives, with Nikon FN-1 microscope equipped with 10x, 20x, and 40x lenses, and with Nikon Crest LFOV Spinning Disk/C2 Confocal equipped with 10x, 20x, and 40x lenses. Images were processed using Imaris software (Bitplane), Fiji (Schindelin et al. 2012 (link)) or NIS-Elements (Nikon).
5
Structural and Functional Mapping of Vascular Connexins
6
Quantifying Neuronal Populations in Confocal Images
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Images were collected on an Olympus FV1000 confocal microscope or a Nikon Widefield microscope. For each quantified value, at least 3 different animals were sampled on 10 consecutive 12 µm sections. Quantification was then manually performed using the Cell Counter plugin of ImageJ. Data shown are expressed as the mean±s.e.m. and are presented in Fig. S1D . Normality tests were applied on datasets and appropriate t-tests were used in order to assess the statistical significance of the data. Significance values are ***P<0.001, **P<0.01, *P<0.05.
7
Confocal Imaging of Brain Tissues and Cultured Neurons
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Coverslips were imaged using a 10x objective for brain tissues and 60x oil objective for cultured neurons on a Nikon FV1000 confocal microscope (Tokyo, Japan). The images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
8
Fluorescent Immunohistochemistry of Spinal Cord
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For fluorescent immunohistochemistry, spinal cord sections or cells were permeabilised using PBST and then incubated overnight at room temperature in primary antibody diluted in PBS-T + 4% goat or donkey serum (rabbit anti-ADAMTS4 Abcam 1:1000; mouse anit-GFAP-Cy3
Sigma 1:500; biotinylated wisteria lectin floribunda WFA, Sigma 1:200; mouse antichondroitin sulphate CS56, Abcam 1:200; goat anti-5HT, Abcam 1:500). After two washes, the cells or sections were then incubated with fluorescent-secondary antibodies (Donkey-antirabbit/goat/mouse Alexa Fluor 488/594, ThermoFisher 1:500; Streptavidin Alexa Fluor488, Sigma 1:200) at room temperature for two hours. Images were captured using an Olympus FV1000 confocal microscope or with a Nikon Eclipse TE2000-U. For fluorescent intensity analyses, images were thresholded to correct for background and the mean pixel intensities
were measured using ImageJ. The same threshold was maintained for all immunostaining groups. For 5HT analysis specifically, the fluorescent intensity from the ventral horns of three sections: 600, 1200 and 1800 µm caudal to the injury measured using ImageJ. The pixel intensity measured at the ventral horns were subtracted from the pixel measured at the dorsal horn within each image.
Sigma 1:500; biotinylated wisteria lectin floribunda WFA, Sigma 1:200; mouse antichondroitin sulphate CS56, Abcam 1:200; goat anti-5HT, Abcam 1:500). After two washes, the cells or sections were then incubated with fluorescent-secondary antibodies (Donkey-antirabbit/goat/mouse Alexa Fluor 488/594, ThermoFisher 1:500; Streptavidin Alexa Fluor488, Sigma 1:200) at room temperature for two hours. Images were captured using an Olympus FV1000 confocal microscope or with a Nikon Eclipse TE2000-U. For fluorescent intensity analyses, images were thresholded to correct for background and the mean pixel intensities
were measured using ImageJ. The same threshold was maintained for all immunostaining groups. For 5HT analysis specifically, the fluorescent intensity from the ventral horns of three sections: 600, 1200 and 1800 µm caudal to the injury measured using ImageJ. The pixel intensity measured at the ventral horns were subtracted from the pixel measured at the dorsal horn within each image.
9
Fluorescence Imaging of Mycobacterium Infection
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Differentiated THP-1 cells (2 × 105 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200). Before confocal microscopy, cells were Hoechst stained (Invitrogen, USA) and treated with an anti-fluorescence quenching agent (Invitrogen, USA). Confocal images were obtained using an Olympus FV1000 confocal microscope (Nikon A1R) and processed with Image J software (NIH, USA). For the image, 80 to 100 cells in each group were analyzed.
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