Cell monolayers cultured on transwell inserts were fixed in cold methanol. For immunofluorescent labeling of TJ and AJ proteins, cell monolayers were incubated with the antibodies mentioned above. Briefly, membranes were blocked with 5% bovine serum albumin in PBS, followed by primary antibody incubation. Membranes were then rinsed three times in PBS, followed by secondary antibody incubation. Furthermore, membranes were rinsed three times in PBS, stained with DAPI, and mounted with an anti-fading mounting medium (Vector Laboratories, Cat. No, H-1000, Burlingame, CA) (26 (link), 30 (link), 31 (link)). Samples were imaged with ×63 objectives using a Leica TCS-SP8-AOBS inverted confocal microscope (Leica Microsystems, Wetzlar, Germany). Images were processed with Adobe Photoshop (RRID:SCR_014199) software.
Tcs sp8 aobs
Manufactured by Leica
Sourced in Germany
The TCS SP8 AOBS is a confocal laser scanning microscope designed for high-performance imaging. It features an Acousto-Optical Beam Splitter (AOBS) for flexible fluorescence excitation and detection. The TCS SP8 AOBS provides advanced imaging capabilities for a wide range of applications.
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Lab products found in correlation
Filmtracer live dead biofilm viability kit, by Thermo Fisher Scientific (2 mentions)
Ab50339, by Abcam (2 mentions)
Cm1950 cryostat, by Leica (2 mentions)
Ab6586, by Abcam (2 mentions)
Aperio imagescope software, by Leica (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Planapo 63x 1.4 na, by Leica (1 mentions)
Dmirb inverted microscope, by Leica (1 mentions)
Hyd detector, by Leica (1 mentions)
Isolectin gs ib4 alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Application suite x las x software, by Leica (1 mentions)
Imagej, by Leica (1 mentions)
Tcs sp2 aobs, by Leica (1 mentions)
Las x navigator, by Leica (1 mentions)
Las x, by Leica (1 mentions)
Spot flex camera, by Nikon (1 mentions)
Tissue studio v 4, by Definiens (1 mentions)
Plan apochromat, by Leica (1 mentions)
Balb cbyj female mice, by Charles River Laboratories (1 mentions)
36 protocols using tcs sp8 aobs
1
Immunofluorescent Labeling of Tight and Adherens Junctions
2
Confocal Microscopy for Subcellular Localization
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Confocal laser scanning microscopy was performed with a LEICA TCS SP8 AOBS inverted confocal microscope (Leica Microsystems). GFP was excited at 488 nm using an argon laser. Fluorescence emission was collected between 500–540 nm. Chloroplasts auto fluorescence was observed at 540 to 700 nm. RFP was excited at 552 nm using an argon laser. For subcellular localization assays with N. benthamiana leaves, confocal laser scanning microscopy was performed 48 h after infiltration. For analysis of plasmolyzed N. benthamiana epidermal cells, leaves infiltrated with A. tumefaciens containing the TaPBS1-GFP construct were mounted in 1 M sucrose before microscopy, as described previously41 (link). Images were processed using Leica Application Suite Advanced Fluorescence (LAS AF) Version 4.3.
3
Visualizing Fungal Spore Germination and Colonization
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To visualize the fungal spore germination events and to determine in planta colonization fluorescent protein expression was studied by an inverted laser-scanning confocal microscope, Leica TCS SP8 AOBS (Leica Microsystems, Exton, PA, Wetzlar, Germany). To study the expression of EGFP in transformed Fox R1, spores were harvested from PDA plates and placed onto a glass slide, and incubated at 25 °C for seven to eight days under high humidity conditions and spores were monitored at regular intervals of times.
4
Complement Protein Treatment on Cortical Slices
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For complement protein treatment, 24 h after the slice preparation (t = 24 h), the medium was supplemented with 1 ng/ml C1q or 1 ng/ml each of C1q and C3 (HyCultBiotech, Uden, The Netherlands) and maintained for 1 or 2 days. Slices were collected (t = 48 or 72 h) on a glass slide, crystals of 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI, Invitrogen, Massachussetts) were placed in the middle of the cortical gray matter using a bamboo fiber under a dissecting microscope, and conserved in a dark room for a further 24 h to allow the diffusion of the dye and the visualization of dendritic segments and dendritic spines. The slices were then rinsed in PB, fixed in 4% PFA for 30 min, mounted with FluorSave™ and examined with a confocal microscope (TCS SP8 AOBS, Leica microsystem). At t = 48 and 72 h, some slices were fixed in 4% PFA and used as immunohistochemical controls to assay the viability using MAP2 and NeuN immunohistochemistry. Cultured slices without complement protein treatment (named not treated) and not cultured slices immediately labeled with DiI (named baseline) were used as internal controls (see Figure 6A for a schematic drawing of the protocol). Unfortunately, not every treatment/time point was verified for each case depending on tissue availability or technical problems (see Table S2 for all the details).
5
Digital Microscopy Image Acquisition and Processing
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Digital microphotographs were acquired using Aperio CS2 technology, and we also used a confocal microscope (Leica TCS SP8 AOBS, Leica Microsystems GmbH, Mannheim, Germany). The images (z-stacks) were acquired with LCS software. The digital confocal images were processed with ImageJ (NIH, https://rsb.info.nih.gov/ij ), Adobe Photoshop and Adobe Illustrator software (Adobe Systems MountainView, CA,USA) and Aperio ImageScope software (Leica Microsystems GmbH, Mannheim, Germany).
6
Digital Microscopic Image Acquisition and Analysis
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Digital photomicrographs were acquired using an Aperio CS2 digitalizing device and a confocal microscope (TCS SP8 AOBS; Leica Microsystems GmbH, Mannheim, Germany). The z-stack images were acquired with LCS software. Digital images were processed with Aperio ImageScope (Leica Microsystems GmbH, Mannheim, Germany), ImageJ (NIH, http://rsb.info.nih.gov/ij ) and Adobe Photoshop and Adobe Illustrator softwares (Adobe Systems Mountain View, CA, USA).
7
Evaluating Acinetobacter baumannii Biofilms
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For evaluation of biofilms, 1000-fold dilutions of overnight A. baumannii cultures were used for seeding into LB media. Biofilms were grown for 2 and 24 h at 37 °C without agitation. After growth in micro-titer plates, biofilms were stained for 2 h by the Filmtracer TM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific). The plate was then placed on the motorized stage of an inverted confocal microscope (TCS SP8 AOBS, Leica Microsystems) at the INRA-MIMA2 imaging platform as described by [45 (link)].
8
Biofilm Viability Assessment Protocol
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For evaluation of biofilms, 1000-fold dilutions of overnight A. baumannii cultures were used for seeding into LB media. Biofilms were grown for 2 h and 24 h at 37 °C without agitation. After growth in micro-titer plates (µclear Greiner BioOne, Les Ulis, France), biofilms were stained for 2 h by the Filmtracer LIVE/DEAD Biofilm Viability Kit (Thermo Fisher Scientific, Les Ulis, France). The plate was then placed on the motorized stage of an inverted confocal microscope (TCS SP8 AOBS, Leica Microsystems, Nanterre, France) at the INRA-MIMA2 imaging platform and results were analyzed as described previously [48 (link)].
9
Confocal Imaging of Dendritic Spines
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Confocal images at different treatments and time points were acquired with a PLANAPO 63x/1.4 NA oil immersion objective by using a TCS SP8 AOBS laser‐scanning confocal microscope (Leica Microsystem) equipped with an Ar/Ar‐Kr 488, 561, and 633 nm diode laser lines and Hybrid detectors. The parameters for acquisition (561 laser line power and Hybrid detector gain) were defined on control conditions and kept constant for all the samples. For the acquisition, we focused on areas where DiI diffused and dendritic spines were clearly visible. Every image included dendritic segments (from 2 to 7) characterized by medium and small thickness, equally represented in the analysis, suggestive of proximal and distal ramifications. The 3D reconstruction of DiI‐filled dendrites was performed using the Simple Neurite Tracer, a dedicated module of Fiji software, and the total dendrite length was measured. The number of dendritic spines was evaluated using the ImageJ program (http://imagej.nih.gov/ij/ ) and the spine density was then calculated. Mean spine density changes at 48/72 h after the two different treatments were calculated (control values set to 1).
10
Visualizing Phenothiazine Dye Accumulation
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The accumulation of phenothiazine dyes in cells was detected by light and confocal microscopies. Firstly, Vero cells were incubated with MB, NMB, TBO, and DMMB (100, 50, 25 and 12.5 µM) for 3 h, 37 °C, 5% CO2 and washed with PBS. The cells were observed in a light microscope and representative pictures of each incubation regimen acquired. For confocal detection of phenothiazinium dyes, Vero cell cultures were incubated with 10 μM of MB, NMB, TBO or DMMB for 30 min, 37 °C, with 5% CO2. For all procedures, non-treated controls were incubated with RPMI under the same conditions. The cultures were washed with PBS and treated with trypsin for 10 min, 37 °C and 5% CO2. The cells were transferred to slides and analyzed by confocal microscopy with excitation/emission wavelengths of 543/600 nm, respectively. The slides were observed in a TCS-SP8 AOBS (Leica Microsystems), using a 63× objective and processed by ImageJ software (version 1.53j, National Institute of Health, USA).
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