BMDM were prepared from C57BL/6 mice and plated on chambered coverslips (Ibidi). Some samples were then infected (multiplicity of infection [MOI], 1,000) with Brucella expressing tdTomato fluorescent protein (Clontech) for 24 h. The high MOI was chosen to increase sensitivity, consistent with our previous studies (18 (link)). Other samples were pulsed with the SIINFEKL peptide (50 μM). Cell samples were fixed in 4% paraformaldehyde and processed for fluorescence confocal microscopy. Cells were stained with a monoclonal antibody to OVA257–264 (OVA residues 257 to 264; SIINFEKL) peptide bound to H2Kb (eBioscience) and then with goat anti-mouse IgG (H+L) Alexa Fluor 488 (Dylight; Thermo Fisher). Imaging was performed at the University of Wisconsin Optical Imaging Core using either a Nikon A1RS confocal microscope or a Leica SP8 3X STED Super-resolution microscope.
Chambered coverslip
Manufactured by Ibidi
Sourced in Germany
Chambered coverslips are a type of laboratory equipment used for cell culture and microscopy. They provide a controlled environment for the growth and observation of cells. Chambered coverslips consist of a glass or plastic base with one or more chambers, allowing for the cultivation of cells under specific conditions.
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Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (2 mentions)
Sp8 gsted, by Leica camera (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
H 2kb, by Thermo Fisher Scientific (1 mentions)
Sp8 3x sted super resolution microscope, by Leica camera (1 mentions)
Mounting medium, by Merck Group (1 mentions)
Sp8 microscope, by Leica camera (1 mentions)
Duolink kit, by Merck Group (1 mentions)
Alexa fluor 594 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Ab104139, by Abcam (1 mentions)
Leica dmi8 inverted widefield microscope, by Leica camera (1 mentions)
Alexa fluor, by Thermo Fisher Scientific (1 mentions)
G1890, by Merck Group (1 mentions)
Insect xpress medium, by Lonza (1 mentions)
Axiocam hrc camera, by Zeiss (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
18 protocols using chambered coverslip
1
Brucella Infection Imaging in Macrophages
2
Proximity Ligation Assay on Cells
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Cells were grown on chambered coverslips (Ibidi), fixed, permeabilised and PLA was carried out using the Duolink kit (Sigma) according to manufacturer’s instructions. Slides were mounted with in situ Mounting Medium (Sigma) containing DAPI. Signals were detected using a confocal SP8 microscope (Leica).
3
Localization of Cholesterol and Organelles in RAW 264.7 Cells
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RAW 264.7 cells were seeded in chambered
coverslips (Ibidi GmbH, Cat# 80826) as per manufacturer’s instructions.
After cells attached, ezFlux (with BODIPY-cholesterol and unlabeled
core) was added in the complete medium (DMEM, 10% FBS) at 0.1 mg per
100,000 cells and incubated for 30 min. The cells were washed with
PBS three times and fixed in 2% buffered formalin for 10 min at 4
°C. The cells were incubated in blocking buffer consisting of
50% fish serum (Aqua block, Eastcoast Bio, Cat#PP82) and 0.2% Triton
X100 in PBS for 30 min at room temperature. Next, the cells were probed
overnight at 4 °C with primary antibodies against early endosomal
antigen (EEA, BD Biosciences, Cat# 610457) or LAMP1 (Developmental
Studies Hybridoma Bank, Cat# 1D4B) at 1:200 dilution in Aqua block
containing 0.1% Triton X100. After five washes with cold PBS, the
cells were incubated with secondary antibodies goat anti-rat IgG Alexa
Fluor 594 (Thermo Fisher A-11007) for LAMP1-treated coverslips and
goat anti-mouse IgG Alexa Fluor Plus 594 (Thermo Fisher A32742) for
EEA coverslips for 1 h at room temperature. In the last 10 min of
incubation, Hoechst 33342 (Thermo Fisher H3570) was added at 10 μg/mL.
The coverslips were washed five times with PBS and imaged immediately
with an Olympus FV1200 confocal microscope.
coverslips (Ibidi GmbH, Cat# 80826) as per manufacturer’s instructions.
After cells attached, ezFlux (with BODIPY-cholesterol and unlabeled
core) was added in the complete medium (DMEM, 10% FBS) at 0.1 mg per
100,000 cells and incubated for 30 min. The cells were washed with
PBS three times and fixed in 2% buffered formalin for 10 min at 4
°C. The cells were incubated in blocking buffer consisting of
50% fish serum (Aqua block, Eastcoast Bio, Cat#PP82) and 0.2% Triton
X100 in PBS for 30 min at room temperature. Next, the cells were probed
overnight at 4 °C with primary antibodies against early endosomal
antigen (EEA, BD Biosciences, Cat# 610457) or LAMP1 (Developmental
Studies Hybridoma Bank, Cat# 1D4B) at 1:200 dilution in Aqua block
containing 0.1% Triton X100. After five washes with cold PBS, the
cells were incubated with secondary antibodies goat anti-rat IgG Alexa
Fluor 594 (Thermo Fisher A-11007) for LAMP1-treated coverslips and
goat anti-mouse IgG Alexa Fluor Plus 594 (Thermo Fisher A32742) for
EEA coverslips for 1 h at room temperature. In the last 10 min of
incubation, Hoechst 33342 (Thermo Fisher H3570) was added at 10 μg/mL.
The coverslips were washed five times with PBS and imaged immediately
with an Olympus FV1200 confocal microscope.
4
Immunofluorescence and Microscopy of FHL2 and p57
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For immunofluorescence HeLa cells were seeded on glass cover slips and grown in 12-well plates. Cells were transfected with FLAG-FHL2 and/or HA-p57-wt encoding plasmids using PEI (polyethylenimine) as transfection reagent31 (link). After 30 hours cells were fixed using 4% paraformaldehyde and permeabilised with 0.1% Triton X-100 (#3051, Roth). After blocking with 0.5% gelatin (G1890, Sigma-Aldrich), samples were incubated with the following primary antibodies: rabbit polyclonal p57 ((C20) sc-1040, Santa Cruz) and monoclonal mouse anti-FLAG (M2 #F3165, Sigma-Aldrich). After washing, cells were incubated with fluorescence labeled antibodies 488 and 555 (Alexa Fluor, Invitrogen). Cells were mounted in a DAPI containing mounting medium (ab104139, Abcam). Analysis was performed on a Leica DMi8 inverted widefield microscope.
For fluorescence microscopy HeLa cells were grown in chambered coverslips (#80826, IBIDI, Planegg, Bavaria, Germany). Cells were transfected with Cherry-FHL2 and/or YFP-p57-wt or p57-Nt/p57-Nt-NLS1 encoding plasmids using PEI or Polyfect as transfection reagent. YFP (yellow fluorescent protein) was excited at 510 nm and mCherry at 550 nm. Analysis was performed on a Leica DMi8 inverted widefield microscope.
For fluorescence microscopy HeLa cells were grown in chambered coverslips (#80826, IBIDI, Planegg, Bavaria, Germany). Cells were transfected with Cherry-FHL2 and/or YFP-p57-wt or p57-Nt/p57-Nt-NLS1 encoding plasmids using PEI or Polyfect as transfection reagent. YFP (yellow fluorescent protein) was excited at 510 nm and mCherry at 550 nm. Analysis was performed on a Leica DMi8 inverted widefield microscope.
5
Membrane Binding of RahU Proteins
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The binding of RahU-mCherry and its single alanine derivative, RahU-Trp94Ala-mCherry, to the membranes of Sf9 insect cells was investigated using fluorescence microscopy. Cells were grown in Insect-XPRESS medium (Lonza, Switzerland) on chambered coverslips (iBidi, Germany) at a final concentration of 300.000 cells/mL. After an overnight incubation at 28°C, cells were washed in a phosphate buffered saline (PBS), fixed with 2% formaldehyde in PBS for 15 min at room temperature and washed again with PBS. For labelling with aforementioned proteins, cells were incubated with 10 µM RahU-mCherry or RahU-Trp94Ala-mCherry for 15 min at room temperature. 10 µM mCherry was used as a negative control. Cell nuclei were labelled with DAPI (4′,6-diamidino-2-phenylindole). Coverslips were examined by a fluorescent microscope AxioImager Z1 supplemented with an ApoTome device enabling optical sectioning, whereas images were acquired using Zeiss AxioCam HRc camera (Carl Zeiss, Germany).
6
Raichu-RhoA FRET Imaging in MDCK Cells
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MDCK cells stably expressing Raichu-RhoA were grown on chambered coverslips (Ibidi). Five days later, images were captured of living cells maintained at 37 °C in MEM without phenol red, supplemented with 0.25% FBS. All images were captured by a multiphoton LSM710 coupled to an AxioObserver microscope (ZEISS) with a 63x/1.2 Water Plan-Apochromat objective lens. Emission ratio imaging was performed with a 458-nm excitation laser/530-nm emission YFP. FRET-RhoAV14 and FRET-RhoAN19 were transfected for 24 h and analyzed to define the range between the active and inactive states of the biosensor. FRET efficiency was analyzed as previously described79 using Fiji software.
7
Cell Proliferation Imaging with EdU Incorporation
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The indicated cells were seeded into chambered coverslips (80826, ibidi) and incubated at 37°C with 5% CO2 condition for 48 h, and then added EdU working solution (10 μM, Yeasen) into culture medium at 37°C with 5% CO2 condition for overnight. After that, the chambers were fixed with 4% paraformaldehyde fix solution for 10 min at room temperature. After following the manufacturer's instruction, DAPI (G1012, Servicebio) were used to counterstain nuclei for 5 min. Confocal microscopes (Leica, Germany) were used to capture digital images.
8
IFN-γ Stimulation of SV40-fibroblasts
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SV40-fibroblasts were plated on chambered coverslips (#80826, iBidi). The following day, they were left unstimulated or were stimulated for the indicated times with 103 IU/mL IFN-γ (Imukin, Horizon Pharma). Cells were fixed by incubation for 15 minutes in 4% formaldehyde in phosphate-buffered saline (PBS), pH 7.4 at 37°C. The cells were then incubated overnight at 4°C with primary antibody (unconjugated, clone D5E4, #8478, Cell Signaling). They were washed three times in PBS, stained by incubation with secondary antibodies for one hour at room temperature (goat anti-rabbit IgG Alexa Fluor 555, #A21429), and left in ProLong Gold with DAPI (#P36931, Thermo Fisher Scientific). Cells were then visualized by confocal microscopy (×63 oil immersion lens, SP8 gSTED, Leica). Images were analyzed with Fiji software.
9
Immunofluorescent Staining of Lung Organoids
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hLOs were cultured as described above in chambered coverslips (Ibidi). hLOs were fixed in 10% neutral buffered formalin for 24 h at 4°C, washed in PBS, and permeabilized and blocked with serum-free protein block (Agilent/Dako) supplemented with 0.5% Triton X-100 for four hours at room temperature. hLOs were incubated with primary antibodies (NKX2.1, HTII-280, KRT5, Supplementary Table 3) overnight at 4°C. hLOs were washed three times in PBS with 0.05% Tween-20 (PBST), then incubated with secondary antibodies (Supplementary Table 3) overnight at 4°C. hLOs were washed three times in PBST, then incubated with NucBlue fixed cell ReadyProbes (Thermo Fisher Scientific) for 10 min at room temperature. Cells were washed three times in PBS, then imaged using a Zeiss LSM 880 confocal microscope.
10
Analyzing Protein Translocation by Confocal Microscopy
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Confocal laser scanning microscopy was used to analyze protein translocation. In preparation, the microsomal fraction was separated from the rest of the translation mixture as described above. 5 µL of the MF were diluted in 15 µL PBS and transferred on chambered Coverslips (ibidi GmbH, Gräfelfing, Germany), The samples were analyzed by confocal laser scanning microscopy using a LSM 510 Meta (Zeiss). Therefore, samples were excited with an argon laser at 488 nm, and the emission signals were recorded with a bandpass filter in the wavelength range from 505 to 550 nm. Photobleaching was performed using an argon laser at 488 nm with 100% laser intensity. After photobleaching pictures were taken each minute for 14 min.
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