For autophagy assay, transfected HCT116 cells expressing fusion protein GFP-LC3 (autophagy marker) were seeded in 6-well plates with coverslips for 24 h. The next day, drugs were added, cells were monitored at 8 and 12 h of treatment and fixed using 3.7% paraformaldehyde (PFA) at room temperature for 30 min and permeabilized with 0.5% Triton X-100 for 3 min. Then, coverslips were rinsed with PBS and were mounted with Vectashield (Vector Laboratories VECTASHIELD® Antifade Mounting Medium with DAPI) and fluorescent images were acquired utilizing the Leica TCS SP8 inverted microscope. All images were processed with Leica Application Suite X.
Tcs sp8 inverted microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP8 inverted microscope is a high-performance imaging system designed for advanced applications in cell biology, developmental biology, and neuroscience research. The microscope features a modular and configurable architecture, allowing it to be tailored to specific experimental requirements. It offers a range of imaging capabilities, including confocal laser scanning, multiphoton excitation, and sensitive fluorescence detection, making it a versatile tool for researchers in life science disciplines.
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Lab products found in correlation
Application suite x, by Leica (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Las x software, by Leica (3 mentions)
Vectashield antifade mounting medium with dapi, by Leica (2 mentions)
Plan apochromat oil objective, by Zeiss (2 mentions)
Plan apochromat oil objective, by Leica (2 mentions)
Lightning mode, by Leica (2 mentions)
Dmi6000, by Leica (2 mentions)
Fixation buffer, by BioLegend (1 mentions)
Mouse anti myc, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti sox2, by Merck Group (1 mentions)
Rabbit anti nestin, by Proteintech (1 mentions)
Rabbit anti tom20, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Rabbit anti tyrosine hydroxylase th, by Merck Group (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Las x core software, by Leica (1 mentions)
17 protocols using tcs sp8 inverted microscope
1
Autophagy Assay in Transfected HCT116 Cells
2
Immunofluorescence Staining of Cellular Markers
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Cells were seeded on 18 mm round coverslips in the 12-well plates, grown for 72 h in the absence or presence of p32-I, incubated with fixation buffer (Biolegend) for 20 min at RT, permeabilized and blocked for 45 min. Fixed cells were incubated in primary antibodies for 3 h at RT, washed and incubated with secondary antibodies for 1 h. Samples were washed and incubated with DAPI (Invitrogen) for 20 min at RT in the dark. Coverslips containing stained samples were mounted onto glass slides using FluorSave ™ reagent (Calbiochem). Primary antibodies used: rabbit anti-TOM20 (Santa Cruz, 1:1000 dilution), mouse anti-MYC (Santa Cruz, 1:250 dilution). Rabbit anti-Nestin (Proteintech, 1:50 dilution), rabbit anti-Sox2 (Millipore, 1:100 dilution). Secondary antibodies used: Alexa Fluor 488 and Alexa Fluor 594 (Invitrogen). Samples were analyzed using Leica TCS SP8 Inverted Microscope.
3
Immunohistochemistry of Drosophila Brain
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The adult Drosophila brains were dissected in PBS, fixed (4% formaldehyde), washed (PBT: PBS + 0.1% Triton X-100), blocked (3% BSA in PBT) for 1 h at room temperature/RT and then incubated in the primary antibody (rabbit anti-Tyrosine Hydroxylase/TH/Sigma, 1:1000 dilution) overnight at 4 °C. On the next day, samples were washed and incubated in the secondary antibody (Cy3-conjugated goat anti-rabbit/Jackson Immunoresearch, 1:500 dilution) for 1.5 h at RT, washed and added the Vectashield mounting media (Vector Laboratories). Stained samples were analyzed using Leica TCS SP8 inverted microscope. Images were collected as z-stacks, each with the size of 1 µm.
4
Yeast-Expressed Fluorescent Protein Microscopy
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Codon-optimized monomeric Green Fluorescent Protein—GFP mUkG1 from soft coral [88 (link)] was synthesized by Genscript and fused to the transporter sequence in the plasmids. Strain EBY_Xyl1 harboring the Cs4130-mUkG1 was grown on YNB without uracil, supplemented with maltose 20 g/L overnight at 30 °C/200 rpm. Yeast cells were examined by confocal fluorescence microscopy (Leica TCS SP8 inverted microscope). The GFP emitted fluorescence was captured by a Hybrid Detector set to the λ-range of 503–530 nm. Differential Interference Contrast (DIC) images were also captured to show yeast morphology. Data were analyzed in LAS X core software (Leica microsystems).
5
Fluorescence Microscopy Imaging Protocols
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Cells were fixed with 3.7% formaldehyde for 10 min at room temp, permeabilized and blocked with 0.5× TBS startblock (37542; Thermo Fisher Scientific) + 0.3% Triton-X100 for 0.5–1 h room temp. Cells were then stained 4°C overnight in antibodies (See Table S6 ) diluted in 0.25× startblock + 0.1% Triton-X100. After 10 min washing in PBS, cells were subjected to 1–2 h staining in secondary antibody diluted in 0.25× startblock + 0.1% Triton-X100 before mounting in Fluoromount-G (Southern Biotech) and imaging. Widefield fluorescence microscopy was performed with a Nikon 80i equipped with 10× (NA 0.45), 20× (NA 0.75), and 60× (NA 1.40) objectives and a CCD camera (Retiga 200R; QImaging). Where indicated, spinning-disk confocal microscopy was performed with a Leica TCS SP8 inverted microscope using a 63× (NA 1.4) oil immersion objective.
Live cell imaging experiments were performed using chambers (1µm-Slide_I_0.4; Ibidi) perfused with a peristaltic pump (Masterflex C/L; Cole Parmer) and pressure dampener in an environmental chamber mounted upon a spinning disc confocal microscope (Leica sp8). For roGFP experiments, 3 × 3 or 5 × 5 fields were imaged within the middle of chambers with a 60× oil immersion objective. For mito-Keima experiments, confocal stacks were acquired using 20× air and 63× oil objectives with Ex/Em of both 405/590 and 552/590.
Live cell imaging experiments were performed using chambers (1µm-Slide_I_0.4; Ibidi) perfused with a peristaltic pump (Masterflex C/L; Cole Parmer) and pressure dampener in an environmental chamber mounted upon a spinning disc confocal microscope (Leica sp8). For roGFP experiments, 3 × 3 or 5 × 5 fields were imaged within the middle of chambers with a 60× oil immersion objective. For mito-Keima experiments, confocal stacks were acquired using 20× air and 63× oil objectives with Ex/Em of both 405/590 and 552/590.
6
Autophagy Regulation by miRNA Modulators
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For autophagy assay, HCT116 and SW480 cells were seeded in six-well plates with coverslips for 24 h. The next day, cells expressing GFP-LC3 (autophagy marker) were treated with 2% FBS and transfected with the mimics of miR-106a, anti-miR, miR-1, and scramble. The cells were monitored at 24 h of treatment. As a positive control were included cells treated only with lipofectamine. Later, they were fixed using 3.7% paraformaldehyde (PFA) at room temperature for 30 min and permeabilized with 0.5% Triton X-100 for 3 min. Then, coverslips were rinsed with PBS and mounted with Vectashield (Vector Laboratories VECTASHIELD® Antifade Mounting Medium with DAPI) and fluorescent images were acquired using the Leica TCS SP8 inverted microscope (objective 40 ×). All images were processed with Leica Application Suite X.
7
Live-Cell Imaging of Spore Germination
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Live-cell imaging of spore germination and CAT fusion was performed in 8-well cell culture chambers. Brightfield light microscopy and differential interference contrast (DIC) microscopy were performed using a Nikon Eclipse TE 2000E inverted microscope with a 60x/1.20 NA water immersion plan apo objective. A Hammamatsu Orca-ER CCD camera and Metamorph software (Universal imaging) was used for image acquisition. Further image analysis was done using Image J software (rsbweb.nih.gov/ij). Confocal microscopy was performed using a Leica TCS SP8 inverted microscope with a white light laser. The white light laser used was set at an excitation wavelength according to the optimal emission peak obtained after a λ-scan for the different fluorophores used. LASAF software was used for image processing. The fluorescent dyes used for labelling organelles are listed in Table 2 and the excitation and emission peaks used for each fluorescent probe are shown in Table 3 . In case of Calcofluor white, a 20 min incubation period at room temperature was required before imaging.
8
Dual-color STED Microscopy of P. berghei Infection
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For dual-color stimulated emission depletion (STED) microscopy, hepatocytes infected with P. berghei sporozoites were fixed 24 hpi with 4% (w/v) paraformaldehyde in PBS followed by 15 min permeabilization in 0.1% Triton-X and 1 h incubation in 10% FCS-PBS blocking buffer. Cells were stained over night by 4°C with 10 µg/ml of anti-LC3 (MBL international, M152–3) and anti-UIS4 (kindly provided by Photini Sinnis) and labeled with 2 µg/ml Oregon Green-488 goat anti-mouse IgG (H+L) (Molecular Probes, Invitrogen, O-6380) and Abberior STAR 440SX goat anti-rabbit IgG (Abberior GmbH, 2–0012–003–4) at room temperature for 1 h. Samples were embedded in Mowiol® 4–88 (Roth, 0713.1) containing 2.5% DABCO® (Roth, 0718.1) antifade.
Microscopy was conducted on the Leica TCS SP8 inverted microscope equipped with gated STED system and a HC PL APO STED WHITE 100x/1.4 oil immersion objective (Leica Microsystems, Wetzlar, Germany). Depletion was performed with a 592-nm depletion laser and images acquired with a time delay of T g = 0.5 ns. Z-Stacks were obtained with increments of 0.22 µm and subsequently deconvolved with the Huygens STED Deconvolution software (Scientific Volume Imaging, Hilversum, Netherlands) and further processed with the image analysis software FIJI.55 (link)
Microscopy was conducted on the Leica TCS SP8 inverted microscope equipped with gated STED system and a HC PL APO STED WHITE 100x/1.4 oil immersion objective (Leica Microsystems, Wetzlar, Germany). Depletion was performed with a 592-nm depletion laser and images acquired with a time delay of T g = 0.5 ns. Z-Stacks were obtained with increments of 0.22 µm and subsequently deconvolved with the Huygens STED Deconvolution software (Scientific Volume Imaging, Hilversum, Netherlands) and further processed with the image analysis software FIJI.55 (link)
9
Time-lapse Imaging of GFP-tagged Proteins
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For time-lapse experiments, HeLa cells expressing the different GFP-tagged proteins were plated on an open µ-Slide with 8 wells (Ibidi, 80826) in complete medium containing 0.5 μM SiR-DNA dye (Spiro Chrome, Thurgau, Switzerland). Images were acquired on a Leica TCS SP8 Inverted Microscope with a 40×/1.30 NA HC Plan APO CS2—OIL DIC objective and argon laser power set at 80%. The Application Suite X software (LAS-X; Leica, Wetzlar, Germany) for multidimensional image acquisition was used. Specimens were maintained at 37 °C and 5% CO2 via a chamber, and z-series of ten 1 µm sections were captured at 2 min intervals. All images were processed using Fiji [22 (link)] to generate maximum intensity projections, to adjust for brightness and contrast, and to create the final movies. The fluorescence intensity values were measured from whole cells (IC) or midbodies (IM) at the different time points indicated using Fiji [22 (link)]. A background intensity value, measured at the same time point and from an identically sized area, was subtracted from each value.
10
Microscopic Observation of Fungal Structures
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For microscopic observation, we exploited a TCS SP8 inverted microscope (Leica) equipped with a 100× objective lens (1.40 numerical aperture), a HyD detector, an FOV scanner, and 488 nm and 561 nm argon lasers for EGFP and FM4-64 fluorescence, respectively. Image data were acquired by using LAS X software (Leica). Kymograph and fluorescence intensity analyses were performed via the respective functions of MetaMorph software (Molecular Devises). For observation culture, approximately 105 conidia of each strain were inoculated with 100 µl of an appropriate medium in a glass-base dish (Iwaki) and incubated at 30 °C for around 20 h. Staining with FM4-64 and Calcofluor White was performed as described previously23 (link). Inhibitor treatments using stocks of nocodazole (NOC; Sigma) and latrunculin B (Lat B; Calbiochem) were carried out as described previously19 (link),47 (link). NOC and Lat B were used at a final concentration of 100 µg/ml and 100 µM from stock solutions at a concentration of 10 mg/ml and 10 mM, respectively, suspended in DMSO.
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