To show the interaction of TMEM106A with lipid raft, a plasmid expressing myc-tagged lipid raft marker CD230 was cotransfected into 293T cells with a plasmid expressing TMEM106A-EGFP. At 16 h posttransfection, cells were fixed for 1 h with 4% paraformaldehyde, washed with PBS, and permeabilized with 0.2% Triton X-100. The cells were stained with anti-myc antibody, TRITC-conjugated secondary antibody and DAPI (Beyotime biotechnology), washed 3 times with PBS, and photographed using a Laser Confocal Microscope (Zeiss LSM700). To show the interaction of TMEM106A with gp160, a plasmid expressing TMEM106A-mCherry was cotransfected into 293T cells with a plasmid expressing gp160-EGFP. To show the interaction of TMEM106A with Gag, a plasmid expressing myc-tagged TMEM106A was cotransfected into 293T cells with pHIV-Gag-iGFP-ΔEnv, which expresses a Gag-GFP fusion protein (Hubner et al., 2007 (link); Micsenyi et al., 2013 (link)). At 16 h posttransfection, cells were fixed for 1 h with 4% paraformaldehyde, washed with PBS 3 times, and permeabilized with 0.2% Triton X-100. Myc-tagged TMEM106A was stained with anti-myc antibody, TRITC-conjugated secondary antibody and DAPI, washed 3 times with PBS, and photographed using a Laser Confocal Microscope (Zeiss LSM700).
Laser confocal microscope
Manufactured by Zeiss
Sourced in Germany, United States
The Zeiss laser confocal microscope is an advanced imaging instrument that utilizes a focused laser beam and a pinhole aperture to capture high-resolution, optical sections of a specimen. It provides a detailed, three-dimensional view of the sample by scanning and collecting light from a single focal plane, allowing for the visualization of intricate structures and details not easily observed with traditional microscopy techniques.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions)
Streptavidin cy3, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Solarbio (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Rabbit asc, by Santa Cruz Biotechnology (3 mentions)
Mitotracker orange, by Thermo Fisher Scientific (3 mentions)
Mitochondrial membrane potential detection kit, by Keygen Biotech (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
T4 dna ligase, by Takara Bio (2 mentions)
Fitc conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Cm1900, by Leica (2 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Inverted microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Paraformaldehyde (pfa), by Sangon (2 mentions)
Ab150073, by Abcam (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
170 protocols using laser confocal microscope
1
TMEM106A Interactions with Lipid Rafts and Viral Proteins
2
LjaMYB12-GFP Fusion Protein Expression
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The coding sequences of LjaMYB12 (without stop codon) were fused to the N-terminus of GFP under the control of CaMV 35S promoter in the p35SGK vector. Primers used for vector construction were listed in Table 1 . The recombinant vector p35SGK-LjaMYB12-GFP was introduced into Agrobacterium tumefaciens EHA105 by chemical transformation. Transient expression of GFP fusion protein in tobacco leaves was conducted through Agrobacterium-mediated transformation as described previously [46 (link)]. Localization of the fluorescence signals was observed using a laser confocal microscope (Zeiss, Oberkochen, Germany).
3
Immunofluorescence Staining of Vimentin
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The cultured NRK-49F cells were fixed in 4% paraformaldehyde at 4 °C for 15 min and then incubated with anti-vimentin (1:200) at 4 °C overnight. The secondary antibodies conjugated to Alexa Fluor 488/594 (Abcam, Cambridge, MA, USA) were added to the slides at room temperature for 30 min, and DAPI was used to stain the nucleus. The image was obtained using a laser confocal microscope (Zeiss, Oberkochen, Germany).
4
Visualization of CMAS and FXR1P Colocalization
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HEK293T and HeLa cells were maintained in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. At 70% confluency, HEK293T and HeLa cells were plated in laser scanning confocal petri dish with basal serum- and antibiotic-free DMEM. The plasmids pEGFP-N1-FXR1 and pDsRed-Monomer-N1-CMAS were single-transfected and co-transfected into HEK293T cells and HeLa cells using Lipofectamine 2000 (35 (link)). After 5 h, the medium was then replaced with complete DMEM containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. After 48 h, the transfected cells were collected and then washed twice with phosphate-buffered saline. Subsequently, 4% paraformaldehyde was used to fix the transfected cells and the nuclei were stained with 4′,6-diamidino-2-phenylindole (Beyotime Institute of Biotechnology, Nantong, China) for 10 min. A laser confocal microscope (Carl Zeiss AG, Oberkochen, Germany) was used to observe the localization and colocalization of CMAS and FXR1P in the cells.
5
Quantifying Reactive Oxygen Levels in H9c2 Cells
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The ROS level of H9c2 cells was detected using a Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions. The reagent used in this kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), has no intrinsic fluorescence. Exposure to ROS in cells results in the oxidation of DCFH-DA to the fluorescent product DCF, which allows the level of ROS in cells to be measured by detecting the fluorescence intensity of DCF. H9c2 cells were incubated with DCFH-DA for 20 min in an incubator (Thermo Fisher Scientific, USA) at 37°C, and the cells were then washed with PBS. Peroxidation levels were investigated and photographed using a laser confocal microscope (Carl Zeiss, Germany) and quantified using ImageJ software.
6
Immunofluorescence Staining of B16BL6 Cells
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B16BL6 cells were seeded on coverslips and grown overnight in complete medium up to 70–80% confluence. Cells were washed thrice with PBS (pH 7.4) and fixed with 2% paraformaldehyde at RT for 5 min. Cells were washed again with PBS and blocked with 3% BSA in PBS for 1 h at RT in humidified chamber. Cells were incubated with primary antibodies for 1 h in humidified chamber, followed by three washes with PBS to remove excess or nonspecifically bound antibody. Cells were further incubated with fluorescent tagged secondary antibodies for 1 h followed by three washes with PBS. Cells incubated only with fluorescent tagged secondary antibody serve as isotype-control. Nuclei were stained with 5 μg/mL of DAPI in PBS for 1-2 min and coverslips were mounted on slides using Vectashield Mounting Medium (Vector Labs). Images were acquired using a Carl Zeiss laser confocal microscope at 63x magnification. Images were analyzed using ImageJ 1.43 software (NIH).
7
Verifying SmbHLH60-SmMYC2 Interaction
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To verify the interaction between SmbHLH60 and SmMYC2, the full-length ORF of SmbHLH60 and SmMYC2 were constructed on pXY106-nYFP and pXY104-cYFP, respectively, and then transformed into A. tumefaciens GV3101. The resuspended Agrobacteria were mixed and injected into N. benthamiana leaves for transient expression. Then N. benthamiana plants were placed in a greenhouse at 26 °C for 48 h, and the yellow fluorescence was observed by laser confocal microscope (Zeiss, Germany) under the excitation of 488 nm lasers previously described [3] (link), [12] (link). This experiment was repeated with three replicates.
8
Immunostaining with QuickBlock™ Reagent
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QuickBlock™ immunostaining blocking reagent (Beyotime Institute of Biotechnology) was used to block nonspecific binding sites, and 3% H2O2 was used to block endogenous peroxidase. Subsequently, the plates were incubated with the primary antibody overnight at 4°C. After washing to remove the primary antibodies, the plates were incubated with a secondary antibody for 2 h at room temperature. A laser confocal microscope (Zeiss AG) was used to visualize target proteins. The primary and secondary antibodies used are listed in Supplementary Table S1 .
9
Mitochondrial Membrane Potential in AML-12 Cells
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The mitochondrial membrane potential of AML-12 cells was detected by JC-1 staining kit. AML-12 cells were fixed in 4% neutral-buffered paraformaldehyde for 15 min. JC-1 dyeing working solution was added and mixed well. The cells were incubated at 37 °C for 20 min. After incubation at 37 °C, the working solution was sucked out and stained with JC-1 buffer (1×), and then washed twice. Image was obtained by laser confocal microscope (Carl Zeiss).
10
Measuring Mitochondrial Potential with JC-1
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Mitochondrial potential was determined with the JC-1 fluorescent probe (Yeasen Biotech) as previously described.[15 (link)] JC-1 forming a monomeric form in low-potential mitochondria produces green fluorescence, while red fluorescence is emitted by the aggregated JC-1 in high-potential mitochondria. After being treated with 200 μmol/L palmitate for 24 h, primary islets or INS-1 cells were incubated in culture media containing JC-1 (10 μmol/L) at 37°C for 30 min. The fluorescence was observed under a laser confocal microscope (Carl Zeiss).
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