Glial cells plated on collagen type 1-coated 8-well culture slides (Corning, Corning, NY, USA) were washed with PBS, fixed with 4% paraformaldehyde for 5 min, and permeabilised with 0.05% Triton X-100 in PBS (PBS-T) for 15 min. The cells were incubated with primary Abs against Cx43, Iba-1, GFAP, NeuN, Nogo-A, or NG2 (detailed information of Abs are listed in Supplementary Table S2 ) in PBS-T with 5% goat serum for 1 h at 37 °C. After rinsing, the cells were incubated with Alexa 488- and 546- or 594-conjugated secondary Abs and 4′,6-diamidino-2-phenylindole (DAPI) for 30 min at 37 °C. Images were captured using a confocal laser microscope system (Nikon A1, Nikon, Tokyo, Japan) with Plan-Apochromat 20 × (0.75 NA) or Plan-Apochromat 10 × (0.45 NA) objective (Nikon) or fluorescence microscope (BZ-X700, Keyence, Osaka, Japan) with Plan-Apochromat 20 × (0.75 NA) or Plan-Fluor 10 × (0.30 NA) objective (Nikon).
Plan apochromat
Manufactured by Nikon
Sourced in Japan
The Plan Apochromat is a high-quality microscope objective lens designed to provide exceptional optical performance. It is a type of apochromatic lens that corrects chromatic and spherical aberrations, resulting in sharp, high-contrast images across a wide field of view. The Plan Apochromat lens is commonly used in various scientific and research applications that require precise, detailed observations.
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Lab products found in correlation
Nis elements software, by Nikon (3 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (2 mentions)
Cd31 fitc, by Thermo Fisher Scientific (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Mouse anti α smooth muscle actin cy3, by Merck Group (2 mentions)
Anti mouse cd68 apc, by BioLegend (2 mentions)
Optiphot 2, by Nikon (2 mentions)
Plan apochromat vc, by Nikon (2 mentions)
Ixonem 897e back illuminated emccd camera, by Oxford Instruments (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Plan apochromat, by Zeiss (2 mentions)
Plan fluor, by Nikon (1 mentions)
Bz x700, by Keyence (1 mentions)
Dfc295, by Leica (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
X cite 120 led boost system, by Zeiss (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
Nis element advanced research software, by Nikon (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
38 protocols using plan apochromat
1
Immunofluorescence Imaging of Glial Cells
2
Quantitative Muscle Histology Analysis
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Paraffin-embedded muscle tissues were sectioned at 10-μm thick, stained with hematoxylin and eosin (H&E), and imaged on a Nikon Eclipse 80i, Nikon Plan Apochromat 20x objective lens (numerical aperture 0.75). For immunofluorescence microscopy, muscle tissue was sectioned at 10-μm-thickness by cryostat. Sections were incubated with CD31-FITC (1:100; Thermo Fisher, 11-0311-85), anti-mouse CD68-APC (1:100; BioLegend, 137008), or anti-mouse α-Smooth Muscle Actin-Cy3 (1:100; MilliporeSigma, C6198), and then mounted with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Thermo Fisher, P36931). Using a Nikon Eclipse 80i inverted microscope with Nikon Plan Apochromat 10x objective lens (numerical aperture 0.3), 6 fluorescence images were acquired in the regions of interest as determined by cellular infiltration using the DAPI staining. Quantification of cell types was expressed as a ratio of each DAPI+ cellular marker (CD68, CD31, SMA) to the total DAPI stained cells in the field. Each individual data point represented one animal and reflected the average of 3–4 images captured from adjacent tissue sections per slide. All subsequent analyses were performed by Image J software(Schneider et al., 2012 (link)).
3
Quantitative Muscle Histology Analysis
4
Multimodal Imaging Techniques for Comprehensive Analysis
5
Fluorescent Labeling of A7r5 Cells
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A7r5 aortic smooth muscle cells (ATCC, Manassas, VA) were cultured on coverslips in DMEM high glucose with 10% fetal calf serum, 1% glutamine, 50 units/mL penicillin, and 50 μg/mL streptomycin. Before experimentation, cells were serum starved for 24 hours to drive them to a differentiated state.33 , 34 To confirm permeation of the peptides into cells, cultured A7r5 cells were either unloaded or loaded for 30 minutes at 37°C with 250 μmol/L N‐WASP CA domain or 100 μmol/L TLN‐VCL fluorescently labeled constructs. For focal adhesion assays, 50 μmol/L TLN‐VCL was used.
Filamentous actin was stained with Alexa Fluor 488 phalloidin (1:3000, Invitrogen, Carlsbad, CA). Cells were examined with an Eclipse TE2000‐E fluorescence microscope (Nikon Instruments) using a Nikon Plan Apochromat oil‐immersion objective and a charge‐coupled device camera (CoolSNAP HQ2, Photometrics, Tucson, AZ). NIS‐Element Advanced Research software (Nikon Instruments) was used to capture images for removal of out‐of‐focus fluorescence blur in tissues by deconvolution of Z‐sections (Richardson‐Lucy algorithm, constrained iterative–maximum likelihood estimation algorithm). Images from control and construct‐loaded samples were obtained at identical microscope and software settings (look‐up tables, offsets, and gains).
Filamentous actin was stained with Alexa Fluor 488 phalloidin (1:3000, Invitrogen, Carlsbad, CA). Cells were examined with an Eclipse TE2000‐E fluorescence microscope (Nikon Instruments) using a Nikon Plan Apochromat oil‐immersion objective and a charge‐coupled device camera (CoolSNAP HQ2, Photometrics, Tucson, AZ). NIS‐Element Advanced Research software (Nikon Instruments) was used to capture images for removal of out‐of‐focus fluorescence blur in tissues by deconvolution of Z‐sections (Richardson‐Lucy algorithm, constrained iterative–maximum likelihood estimation algorithm). Images from control and construct‐loaded samples were obtained at identical microscope and software settings (look‐up tables, offsets, and gains).
6
cGAS Immunofluorescence Imaging Protocol
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Cells on coverslips were washed in 1xPBS, swollen in hypotonic solution (85.5 mM NaCl and 5 mM MgCl2) for 5 min and fixed with 4% paraformaldehyde for 10 min, before permeabilizing with 4% paraformaldehyde + 0.03% SDS. Cells were rinsed with 1xPBS + 0.4% Photo-Flo 200 (P7417-1PT, Sigma) and blocked with blocking buffer (1% goat serum, 0.3% BSA, 0.005% Triton X-100 in PBS). Then, cells were incubated with rabbit monoclonal antibody against cGAS (1:200, 15102, Cell Signaling) in blocking buffer overnight at room temperature. The next day, cells were washed and incubated with Alexa Fluor 488-conjugated secondary antibodies (1:400, A11008, Thermo) for 1 h at room temperature. Dried coverslips were mounted on microscope slides using ProLong Gold antifade reagent with DAPI (P36935, Thermo). Images were acquired using a Nikon Plan Apochromat 60 × 1.4 NA oil immersion lens on the Dragonfly imaging system (Andor Technologies, Belfast UK) equipped with 405 and 488 nm lasers for visualizing DAPI and cGAS immunostained with an Alexa Fluor 488-conjugated antibody, respectively. The Dragonfly is built on a Nikon Ti2-E microscope body with the Nikon Perfect Focus System (Nikon Instruments, Japan). Data was collected using the Spinning Disk 40 µm pinhole mode of the Dragonfly on a Zyla 4.2 sCMOS camera using Fusion v2.0.0.15 software (Andor Technologies).
7
Mitochondria Visualization and Analysis by Confocal Microscopy
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Mitochondria fluorescence was studied by laser scanning confocal microscopy, using a Bio-Rad MRC-600 confocal microscope (Bio-Rad, Hemel Hempstead, UK) equipped with a 25 mW argon laser. The scanning head was coupled with an upright epifluorescence microscope Nikon Optiphot-2 (Nikon, Tokyo, Japan) with a 60× oil immersion objective Nikon Planapochromat (N.A. = 1.4). The fluorescence was excited at 488 nm and the emission was collected through a long pass filter above 515 nm. High sensitivity photon counting detection was used to minimize the excitation power (0.1 mW at the entry of the optical head) and preserve cell viability.
The cells were plated in 35 mm Ø Petri dishes at density of 6 × 104 cells/well and 24 h after seeding the medium was removed, the cells were washed twice with phosphate buffer saline (PBS) and incubated for 10 min in 1 μM rhodamine 123 (R123) solution at 37 °C and 5% CO2. After incubation, the cells were rinsed twice with PBS and few microliters of PBS were left in the Petri dish to avoid cell drying. A coverslip was placed over the cells that were immediately imaged by confocal microscope.
To measure mitochondrial interconnectivity and elongation from confocal microscope images we used the macro designed by Dagda and colleagues [45 (link)].
The cells were plated in 35 mm Ø Petri dishes at density of 6 × 104 cells/well and 24 h after seeding the medium was removed, the cells were washed twice with phosphate buffer saline (PBS) and incubated for 10 min in 1 μM rhodamine 123 (R123) solution at 37 °C and 5% CO2. After incubation, the cells were rinsed twice with PBS and few microliters of PBS were left in the Petri dish to avoid cell drying. A coverslip was placed over the cells that were immediately imaged by confocal microscope.
To measure mitochondrial interconnectivity and elongation from confocal microscope images we used the macro designed by Dagda and colleagues [45 (link)].
8
Mitochondrial Fluorescence Imaging in Cells
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Mitochondria fluorescence in living cells was studied by laser scanning confocal microscopy, using a BioRad MRC600 confocal microscope equipped with a 25mW argon laser (BioRad, Hemel Hempstead, UK). The scanning head was coupled with an upright epifluorescence microscope Nikon Optiphot2 (Nikon, Tokyo, Japan) with a 60x oil immersion objective Nikon Planapochromat (N.A. = 1.4).
For R123 staining, the cells were plated in 35mm Petri dishes at a density of 6 x 10 4 cells and left to grow for 24 hours in culture medium. Then CdCl 2 was added to the medium to a final concentration of 1 or 4 μM CdCl 2 . After 24 hours, the medium was removed, cells were washed twice with phosphate buffer saline (PBS) and incubated for 10 min in 1 μM Rhodamine 123 (R123) solution at 37 °C and 5% CO 2 . After incubation, the cells were rinsed twice with PBS and few microliters of PBS were left in the Petri dish to avoid cell drying. A coverslip was placed over the cells that were immediately imaged by confocal microscope.
R123 fluorescence was excited at 488 nm and the emission collected through a long pass filter above 515 nm. High sensitivity photon counting detection was used to minimize the excitation power (0.1mW at the entry of the optical head) and preserve cell viability.
For R123 staining, the cells were plated in 35mm Petri dishes at a density of 6 x 10 4 cells and left to grow for 24 hours in culture medium. Then CdCl 2 was added to the medium to a final concentration of 1 or 4 μM CdCl 2 . After 24 hours, the medium was removed, cells were washed twice with phosphate buffer saline (PBS) and incubated for 10 min in 1 μM Rhodamine 123 (R123) solution at 37 °C and 5% CO 2 . After incubation, the cells were rinsed twice with PBS and few microliters of PBS were left in the Petri dish to avoid cell drying. A coverslip was placed over the cells that were immediately imaged by confocal microscope.
R123 fluorescence was excited at 488 nm and the emission collected through a long pass filter above 515 nm. High sensitivity photon counting detection was used to minimize the excitation power (0.1mW at the entry of the optical head) and preserve cell viability.
9
Immunofluorescence Staining of Adherent Cells
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The cells were grown on a poly-l -lysine–coated coverslip. For immunostaining, the cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4) for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. The fixed cells were blocked with 3% bovine serum albumin (BSA)/PBS for 1 hour at room temperature. Then, the cells were incubated with the primary antibodies diluted with 3% BSA/PBS for 1 hour in a humidity box, stained with Alexa Fluor–conjugated secondary antibodies for 1 hour, counterstained with DAPI for 5 min, and mounted with VECTASHIELD (Vector Laboratories). Images were acquired using a BioRevo microscope (BZ-9000; Keyence) equipped with a 40× objective lens [Plan Apochromat, numerical aperture (NA) 0.95, Nikon] and a 100× oil objective lens (Plan Apochromat VC, NA 1.4). Cellular counting and image cropping were performed using ImageJ.
10
Immunofluorescent Imaging using Nikon E600
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Immunofluorescent imaging was performed using Cy3 HYQ, FITC HYQ, and a fluorescence microscope (E600; Nikon) with Plan Apochromat objective lenses. Images were acquired using a digital camera (DXM1200F; Nikon) and Act-1 software version 2.63 (Nikon). Images were composed in Photoshop CS6 (Adobe).
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