Third instar larvae were dissected, fixed and stained as previously described (47 (link)). Briefly, larvae or adult were dissected in iced-cold phosphate buffered saline (PBS) (Lonza, #17–516F). Dissected larvae were fixed in 4% formaldehyde, washed 3× in PBS, then washed 3× in 0.1% PBST (0.1% Triton X-100 in PBS) and incubated overnight with primary antibodies (mouse anti-lamin, DSHB, 1:200, mouse anti-mCherry 1:100 rabbit anti-TDP-43, Proteintech 10782-2-AP, 1:200; mouse anti-mono and poly ubiquitinylated conjugates, Enzo Life Sciences BML-PW8810, 1:100; rat anti-phospho Ser409/410 TDP-43, EMD Millipore clone 1D3, 1:50) in 0.1% PBST. Then, Larvae were washed 3× in 0.1% PBST (0.1% Triton X-100 in PBS) and incubated for 2 hours in secondary antibodies (anti-rabbit Alexa Flour 568, Invitrogen, # 651727, 1:100; anti-mouse Alexa Flour 647, Invitrogen, # 28181, 1:100; anti-rabbit Alexa Flour 405, Life Technologies, # 157554, 1:100; anti-rabbit Alexa Flour 647, Life Technologies, # 1660844, 1:100; anti-rat Alexa Fluor 488, Invitrogen, #A-11006), and mounted using Fluoroshield (SIGMA, #F6182). Slides were visualized using a Nikon A1 laser-scanning confocal microscope system utilizing 60X oil immersion objectives (CFI Plan Apo Lambda 60X Oil, Nikon).
Alexa flour 647
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647 is a fluorescent dye produced by Thermo Fisher Scientific. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the far-red region of the visible spectrum. Alexa Fluor 647 can be used to label a variety of biological molecules, such as proteins, nucleic acids, and other biomolecules, for various applications in life science research and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti mouse antibody, by Jackson ImmunoResearch (17 mentions)
Oil na1.4 objective, by Nikon (10 mentions)
Hap043396, by Merck Group (5 mentions)
Sc 126, by Santa Cruz Biotechnology (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Sc 101199, by Santa Cruz Biotechnology (3 mentions)
Hap035000, by Merck Group (3 mentions)
Alexa flour 488, by Thermo Fisher Scientific (3 mentions)
Facsaria 2, by BD (3 mentions)
Alexa flour 568, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Cfi plan apo lambda 60x oil, by Nikon (2 mentions)
Fluoroshield, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Mlc 400 monolithic laser combiner, by Keysight (2 mentions)
Brilliant violet 421, by BioLegend (2 mentions)
Live dead stain, by Thermo Fisher Scientific (2 mentions)
Cell surface markers, by BD (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
46 protocols using alexa flour 647
1
Immunostaining of Drosophila Larvae
2
Visualizing Neutrophil Extracellular Traps
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For NET visualization with antibodies, the primary anti-human MPO antibody (DakoCytomation- A0398) was diluted into 10% FBS in PBS (1/500). 30 µL was added to adhered neutrophils, incubated (30 min, 37°C) and washed twice with sterile PBS. 40 µL of the secondary anti-rabbit Cy 5 antibody (Jackson ImmunoResearch 60354) (1/500 dilution) was added. After 15 min incubation in the dark, cover slips were washed twice with PBS, and prepared with mounting media. Anti-DNA and anti-histone antibodies where obtained from Dr. Marvin Fritzler. Either the anti-DNA (1:10) or anti-histone (1:500) antibodies [36 (link), 37 (link)] were added as described above. The anti-human secondary antibodies with Alexa Flour 647 (Invitrogen A21445, 1/500) were added to cover slips and mounted as described above. Images of human NETs were acquired using the Leica DMI 4000B inverted microscope equipped with ORCA R2 digital camera and Metamorph software for image acquisition using the 63X or 100X objectives. The following excitation and emission filters were used for blue fluorescence (Ex 390/40; Em 455/50), red fluorescence (Ex 555/25; Em 605/52), far red fluorescence (Ex 645/30; Em 705/72) and green fluorescence (Ex 490/20; Em 525/36). Images were formatted and analyzed using the Imaris 7.0.0 imaging software. All images shown are representative of at least three experiments.
3
Multiparametric Analysis of DNA Damage Response
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Primary antibodies: XAB2 (abcam, ab129487, dilution 1:1000), RAD51 (Merck, Ab-1, PC130, dilution 1:1000), γH2AX (Millipore, Cat. No. 05-636, Clone JBW301, 1:1000), 53BP1 (abcam, ab36823, 1:1000), pS1778-53BP1 (Cell signalling, 26755, 1:1000), pS25/29-53BP1 (Cell signalling, 2674S, 1:1000), pRPA32 (S4/S8) (Bethyl Laboratories, A300-245A, 1:1000), Ku80 (abcam, ab80592, 1:1000), BrdU (GE Healthcare, RPN202, dilution 1:1000), PCNA (Chromotek, 16D10-100, 1:500). Secondary antibodies: Alexa Flour 647 (Invitrogen, A-21235, 1:500), Alexa Flour 555 (Invitrogen, A-21422, 1:500), Alexa Flour 488 (Invitrogen, A-11001, 1:500), Alexa Fluor 568 (Invitrogen, A-11077, 1:500).
4
Immunofluorescence Analysis of USP1 and ERα
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit Anti-USP1 (SAB1406575, Sigma) rabbit antibody and mouse anti-ERα monoclonal antibody (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
5
Immunofluorescence Assay for SHARPIN
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The immunofluorescence assay was described in detail in our previous study. MCF-7 cells were treated with estradiol or vehicle for 30 min before being fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-SHARPIN polyclonal antibody and mouse anti-monoclonal antibodies were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson Immuno-Research, West Grove, PA).
6
Histological Analysis of Kidney Injury
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After euthanasia, mice were perfused with PBS via intracardiac puncture and organs were collected in 10% neutral buffered formalin. The tissues were further processed and embedded in paraffin. H&E staining was conducted as described previously.38 For IgG deposition in the glomerulus, sections were deparaffinized with histoclear and hydrated using a series of graded ethanol and finally transferred to 1X TBS (Tris‐buffered saline). Antigen retrieval was performed using a heated sodium citrate buffer (10 mM sodium citrate, pH 6.0). For detecting IgG immune complex in kidney, the sections were blocked in 3% FBS in PBS followed by incubation with anti‐mouse IgG conjugated to Alexa flour 647 (Cat# A21236, Invitrogen) for 1 h. After three washes with TBS containing 0.5% tween 20, images were acquired on a fluorescence microscope followed by data analysis in ImageJ. Kidney injury scoring was conducted in H&E and PAS‐stained sections as described previously.39 , 40 Briefly, the analysis included examination of the percentage of tissue demonstrating infiltrating leukocytes, tubular dilatation, cast formation, and loss of tubular brush border with an arbitrary scoring system as follows: 0 = none; 1 = <10%; 2 = 11%–25%; 3 = 26%–45%; 4: 46%–75%; 5 = >75%.
7
Immunofluorescence Staining of MDAMB231 Cells
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4% paraformaldehyde was used to fix MDAMB231 cells for 15 min, 0.25% Triton X-100 to permeabilize them for 15 min, and 3% BSA to block them for 1 h at room temperature. The rabbit anti-RNF31 antibody (ab125189, Abcam, 1:400), mouse anti-YAP antibody (SC-101199, 1:200) and mouse anti-PD-L1 antibody (1:400, 66,248–1-Ig, Proteintech) were used. The Alexa Flour 647 (Invitrogen) anti-mouse or Alexa Flour 488 anti-rabbit secondary antibodies (Invitrogen). The nuclei were stained with DAPI (Beyotime). Samples with only secondary antibodies and no primary antibodies were used as negative controls. Images were captured with a Nikon A+ laser scanning confocal system.
8
Immunofluorescence Imaging of SHARPIN and YAP
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EC109 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-SHARPIN polyclonal antibody (Ab125188, Abcam) and mouse anti-YAP monoclonal antibodies (SC-101199, Santa Cruz) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
9
Immunofluorescence Imaging of RNF187 and YAP
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EC109 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-RNF187 polyclonal antibody (HAP030098, Sigma, 1/50) and mouse anti-YAP monoclonal antibodies (SC-101199, Santa Cruz, 1/50) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60× oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
10
Immunofluorescent Localization of TRIM3 and P53
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-TRIM3 polyclonal antibody (HAP043396, Sigma) and mouse anti-P53 monoclonal antibodies (SC-126, Santa Cruz) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A + laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
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