Cellular morphology was visualized at week1 and week2 using fluorescence microscopy. Briefly, cells and cell-laden constructs were fixed with 4% paraformaldehyde (PFA) in PBS (pH7.4) for 10 min at room temperature (RT). After rinsing with PBS for three times, the samples were placed in a permeabilization solution with 0.1% (v/v) Triton X-100 for 10 min and rinsed again with fresh PBS for three times. The cells and constructs were then blocked with 1% BSA in PBS for 1 hour at RT. Immunostaining with primary antibody rabbit monoclonal FSP1 (Abcam, Cambridge, UK) (1:400) and Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Life technologies, Carlsbad, CA) (1:1000) was performed at 4 °C with gentle shaking for overnight, then counterstained with Phalloidin-Atto 488 and Hoechst 33258 (Life technologies, Carlsbad, CA) to visualize the f-actin and nuclei, respectively. The cells and cell-laden constructs were visualized using a Zeiss LSM 700 laser confocal microscope (Carl Zeiss Micro-Imaging GmbH, Germany).
Alexa fluor 647 conjugated donkey anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647-conjugated donkey anti-rabbit IgG is a secondary antibody reagent that can be used to detect and visualize rabbit primary antibodies in various immunodetection applications, such as Western blotting, immunohistochemistry, and flow cytometry. The antibody is conjugated to the Alexa Fluor 647 fluorescent dye, which has excitation and emission wavelengths suitable for detection in the far-red/near-infrared region of the spectrum.
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Lab products found in correlation
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (5 mentions)
Prolong gold, by Thermo Fisher Scientific (5 mentions)
Tcs sp5 spectral confocal microscope, by Leica (4 mentions)
Rabbit anti γ tubulin, by Merck Group (4 mentions)
Alexa fluor 546 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (3 mentions)
A31573, by Thermo Fisher Scientific (2 mentions)
Axiovert 200m microscope, by Zeiss (2 mentions)
Zen 2010, by Zeiss (2 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated donkey anti goat igg, by Thermo Fisher Scientific (2 mentions)
Anti vβ2tcr mab, by Beckman Coulter (2 mentions)
Alexa fluor 546 conjugated donkey anti goat igg, by Thermo Fisher Scientific (2 mentions)
Lsm 700 laser confocal microscope, by Zeiss (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Phalloidin atto 488, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin r415, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin a12379, by Thermo Fisher Scientific (1 mentions)
Anti gfp a6455, by Thermo Fisher Scientific (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
Anti rac1, by Cytoskeleton (1 mentions)
23 protocols using alexa fluor 647 conjugated donkey anti rabbit igg
1
Cellular Morphology Visualization by Fluorescence Microscopy
2
Antibodies for Western Blotting and Immunostaining
3
cFos Immunohistochemistry in Mouse Brain
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For cFos immunohistochemistry (IHC), mice were deeply anesthetized with pentobarbital (150 mg/kg intraperitoneally, Streuli Pharma, Switzerland) and perfused trans-cardially (4.0% paraformaldehyde, 1X PBS, pH 7.4). Brains were removed, post-fixed (4% PFA overnight), and cryoprotected (30% sucrose, 1X PBS, 4 °C, 48 h). They were then frozen and 40 μm coronal sections were cut with a sliding cryostat (Leica Microsystems, Germany).
Subsequently, free floating sections were incubated in blocking solution (1% BSA, 1X PBS, 0.3% TrytonX100) at room temperature for 1 h, followed by incubation with rabbit anti-cFos antibody (1:5000, Synaptic System, Germany, #226 003) in blocking buffer (1% BSA, 1X PBS, 0.1% TrytonX100) overnight at 4 °C under constant shaking. Sections were washed extensively with PBS Tryton 0.1% and then exposed to the secondary antibody (Alexa Fluor 647-conjugated donkey anti-rabbit IgG, Life Technologies, USA) in blocking buffer at room temperature for 2 h. After extensive washing, the sections were incubated with Hoechst (Life Technologies, USA) at 1:1000 in PBS at room temperature for 5 min. Slices were washed extensively with PBS and mounted on superfrost glass slides (ThermoScientific, USA) with Fluoromount mounting medium (SouthernBiotech, USA). Images were acquired on a virtual slide microscope (VS120, Olympus, Japan) with a 10 × objective.
Subsequently, free floating sections were incubated in blocking solution (1% BSA, 1X PBS, 0.3% TrytonX100) at room temperature for 1 h, followed by incubation with rabbit anti-cFos antibody (1:5000, Synaptic System, Germany, #226 003) in blocking buffer (1% BSA, 1X PBS, 0.1% TrytonX100) overnight at 4 °C under constant shaking. Sections were washed extensively with PBS Tryton 0.1% and then exposed to the secondary antibody (Alexa Fluor 647-conjugated donkey anti-rabbit IgG, Life Technologies, USA) in blocking buffer at room temperature for 2 h. After extensive washing, the sections were incubated with Hoechst (Life Technologies, USA) at 1:1000 in PBS at room temperature for 5 min. Slices were washed extensively with PBS and mounted on superfrost glass slides (ThermoScientific, USA) with Fluoromount mounting medium (SouthernBiotech, USA). Images were acquired on a virtual slide microscope (VS120, Olympus, Japan) with a 10 × objective.
4
TRAF6 and GFAP Expression Analysis
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The sections were treated with 5% goat serum and 0.1% Triton X-100 to block nonspeci c binding. Then, the sections were incubated overnight at 4 °C with a rabbit anti-TRAF6 antibody (Bioss Technologic Inc., China) and an anti-GFAP antibody (mouse monoclonal, 1:600; CST, USA). After washing with PBS, the sections were incubated with a mixture of Alexa Fluor 647-conjugated donkey anti-rabbit IgG and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500; Life Technologies) for 30 min at 37 °C. To identify the nuclei of the cells, the sections were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 5 min. After staining, the sections were mounted with Ultramount (DAKO) and photographed with a confocal uorescence microscope (TCS-TIV; Leica, Nussloch, Germany).
5
Confocal Microscopy of Transfected Cells
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Confocal microscopy experiments were described previously (Choe and Kirkegaard, 2004 (link)). In brief, the transfected cells were fixed with 3% paraformaldehyde (PFA), washed with PBS and permeabilized with 0.2% Triton X-100/PBS. Anti-flag and Anti-HA antibodies were used as the primary antibodies (1:1000 dilution), Alexa Fluor 488-conjugated Donkey anti-mouse IgG and Alexa Fluor 647-conjugated Donkey anti-rabbit IgG were used as secondary antibodies (1:500 dilution; Molecular Probes, abcam). DAPI dyes (Beyotime Institute of technology, China) was used for cell nucleus stains. The cells were examined and images were captured using 100x objectives with a confocal microscope (Leica SP8). The images were refined and figures were generated using Adobe Photoshop software (Adobe Systems, San Jose, CA).
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Immunofluorescence Staining of ATP5B and TAX1BP1
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The cells were seeded on a sterilized coverslip in a 24-well plate for overnight. After treatment, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.2% Triton X-100 for 10 min at room temperature, and then blocked with blocking buffer (2% BSA and 0.3 M glycine in PBS) for 1 h at room temperature. The cells were then incubated with primary antibodies diluted in blocking buffer for overnight at 4 °C. After washing three times with PBS, cells were incubated with Alexa Fluor-conjugated secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR, USA) diluted in blocking buffer for 1 h at room temperature. After washing with PBS, cells were sealed with nail polish. Fluorescence images were taken under a Nikon A1R + /A1 Confocal Microscope (Nikon, Tokyo, Japan). The antibodies used in this study are as follows: ATP5B (1:25, Santa Cruz Biotechnology, #sc-166462), TAX1BP1(1:50, Proteintech, #14424-1-AP), Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:500, Molecular Probes, #A-21207) and Alexa Fluor 647-conjugated donkey anti-rabbit IgG (1:500, Molecular Probes, #A-21245).
7
Imaging Cellular Structures with Confocal Microscopy
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Two days after transfection, HeLa cells on 35-mm glass-bottom dishes were imaged using an inverted laser scanning confocal microscopy system (Olympus FV3000) equipped with a ×60 water objective lens (Olympus, UPlanApo ×60/1.2 NA). The size of the confocal aperture was 1 Airy disk.
In experiments of microtubule, filamentous actin and plasma membrane localizations (Supplementary Fig.9a–c ), cells were live imaged. When StayGold was localized to the Golgi apparatus (Supplementary Fig. 9d ), cells were fixed with 4% PFA at room temperature for 5 minutes. Fixed cells were permeabilized in 0.1% Triton X-100/PBS for 30 minutes and then reacted with rabbit anti-GM130 polyclonal antibody (PM061, MBL, 1:500 dilution) for 1 hour and Alexa Fluor 647-conjugated donkey anti-rabbit IgG (A31573, Thermo Fisher Scientific, 1:500 dilution) for 1 hour. In addition, cell samples were stained with DAPI (D523, Fuji Film, 1:1,000 dilution). Confocal images were acquired every 1 μm along the z-axis to create z-stacks (20 slices).
In experiments of microtubule, filamentous actin and plasma membrane localizations (Supplementary Fig.
8
GPIHBP1 Binding and LPL Localization
9
Immunofluorescence Staining of HUVECs
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HUVECs were washed two times with phosphate-buffered saline (PBS) and fixed for 10 min at room temperature with 4% paraformaldehyde in PBS. After three washes with PBS, the cells were permeabilized for 10 min at room temperature with 0.2% Triton X-100 in PBS and blocked with 5% bovine serum albumin for 1 h at room temperature. Cells were then incubated overnight at 4°C room temperature with the primary Ab diluted in blocking solution. The following primary antibodies were used: H3K9ac (Cell Signaling 9649; 1:400), H3K27me3 (Cell Signaling 9733; 1:1000), H3K9me3 (Cell Signaling 13969; 1:100), H3K4me3 (Cell Signaling 9751; 1:400), phospho-histone H2A.X (Ser139; Cell Signaling 9718; 1:400), CD29 Integrin beta 1 (Thermo Scientific; 14-0299-82; 1:100), lamin A/C (Santa Cruz Biotechnology; sc-7292; 1:100), or VE-cadherin (Cell Signaling Technology; D87F2; 1:250). Three more washes with PBS were then followed by incubation with the secondary Ab (Alexa Fluor 647–conjugated donkey anti-rabbit IgG; Thermo Fisher) and stained with rhodamine phalloidin (cat. # PHDR1; Cytoskeleton) for 45 min followed by three additional PBS washes. Samples were stained with Hoechst 33342 (Thermo Fisher) and mounted with ibidi mounting medium (ibidi; cat. #50001; Germany).
10
Immunofluorescence Imaging of Cardiac Organelles
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Fresh heart tissues were fixed in 4% paraformaldehyde, transferred to 18–10% sucrose in PBS, and embedded in OCT. Samples were sectioned at 8 μm, air-dried and stored at −80°C until used. Frozen slides were then thawed, permeabilized, blocked with 3% donkey serum (Jackson ImmunoResearch catalog number 017-000-121) in PBS, and subjected to staining by a rat monoclonal anti-Lamp1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number sc-1992; 1:300) or a rabbit monoclonal anti-Mfn2 (Cell Signaling, Danvers, MA, catalog number 9482; 1:100 at room temperature, 1 h). Upon completion of secondary antibody incubation with Alexa Fluor 448 conjugated donkey anti-rat IgG (Thermo Scientific; catalog number A-21208; 1:300) or Alexa Fluor 647 conjugated donkey anti-rabbit IgG (Thermo Scientific; catalog number A-31573; 1:300), the slides were washed, sealed with DAPI/antifade mounting solution (Thermo Fisher Scientific, Rockford, IL; catalog number 36931), and examined. Images were acquired with a LSM 510 confocal microscope equipped with an Axio Observer Z1 motorized inverted microscope and Zen software (Carl Zeiss Microscopy) and analyzed offline with Imaris software (version 9.5, Bitplane).
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