Larvae were rinsed with 1% PBTx, (1% Triton X-100 in PBS), permeated in 0.24% trypsin in PBS and blocked for 3 hours in block buffer (10% normal goat serum (NGS) in 1% PBTx). Samples were incubated with anti-L-plastin [1:500 (V/V) dilution] in antibody buffer (PBTx containing 1% (V/V) NGS and 1% (W/V) BSA) overnight at RT. Samples were washed with PBTx, incubated for 1 hour in block buffer and stained with an Alexa-Fluor-647 goat-anti-rabbit antibody (Invitrogen A21070, 1:400), overnight at 4°C.
Alexa fluor 647 goat anti rabbit antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa-Fluor-647 goat-anti-rabbit antibody is a secondary antibody conjugated with the Alexa Fluor 647 fluorescent dye. It is designed to bind to primary rabbit antibodies and can be used for detection and visualization in various immunoassay techniques, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Thiostrepton, by Merck Group (1 mentions)
Carboplatin, by Selleck Chemicals (1 mentions)
Cleaved parp antibody, by Abcam (1 mentions)
Foxm1 antibody, by Cell Signaling Technology (1 mentions)
Goat anti mouse antibody, by Merck Group (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Alexa fluor 568 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Eight well chamber slide, by Thermo Fisher Scientific (1 mentions)
H 1200, by Vector Laboratories (1 mentions)
Fluoview fv3000, by Olympus (1 mentions)
Al177, by Adipogen (1 mentions)
Peg cat, by Merck Group (1 mentions)
Peg sod, by Merck Group (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Anti ha tag, by Proteintech (1 mentions)
Dead end fluorometric tunnel system, by Promega (1 mentions)
Basic glial cells nucleofector kit, by Lonza (1 mentions)
6 protocols using alexa fluor 647 goat anti rabbit antibody
1
Immunohistochemical Staining of L-Plastin
2
Immunohistochemical and Immunofluorescence Analyses
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Carboplatin and olaparib were purchased from Selleck Chemicals (Houston, TX, USA). Thiostrepton was obtained from Sigma-Aldrich. Pan-cytokeratin antibody was acquired from Bio Genex Laboratories (San Ramon, CA, USA), Ki67-antibody from DCS (Hamburg, Germany), cleaved-PARP-antibody from Abcam (Cambridge, UK) and FOXM1-antibody from Cell Signaling Technology (Danvers, MA, USA). Antibody against CD3 was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA), antibody against CD8 from Cell Signaling Technology and antibody against CD68 from Agilent. Secondary antibodies for immunofluorescence Alexa Fluor 568 goat anti-mouse antibody and Alexa Fluor 647 goat anti-rabbit antibody were obtained from Invitrogen (Carlsbad, CA, USA). Secondary biotinylated goat anti-rat antibody was purchased from Vector Laboratories (Burlingame, CA, USA) and goat anti-mouse antibody was acquired from Sigma Aldrich for immunohistochemical analysis.
3
Fluorescent Imaging of Macrophage Autophagy and Secretion
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BMMs (1 × 105) were seeded in eight-well chamber slides (Thermo Fisher Scientific). After the treatment with different stimuli, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 for 15 min, followed by blocking with 1% bovine serum albumin and 0.1% Triton X-100 in phosphate-buffered saline (PBS) at 37 °C for 1 h. The samples were incubated with anti-ASC antibody (AL177, Adipogen), anti-ALIX antibody (634501, Biolegend), anti-CHMP4B antibody (13683-1-AP, Proteintech), and anti-LAMP1 antibody (1D4B, Developmental Studies Hybridoma Bank) in the blocking buffer overnight at 4 °C, followed by the incubation with Alexa Fluor 647 goat anti-rabbit antibody (A-21244, Thermo Fisher Scientific), Alexa Fluor 488 goat anti-rabbit antibody (A-11008, Thermo Fisher Scientific), or Alexa Fluor 568 goat anti-rat antibody (A-11077, Thermo Fisher Scientific) for 2 h at room temperature. Nuclei were stained with DAPI (H-1200, Vector Laboratories). To evaluate LAP formation, BMMs, generated from GFP-LC3 transgenic mice, were incubated with zymosan at a ratio of 8:1 (particle/cell), then GFP-LC3 puncta were detected. Images were acquired using a laser scanning confocal fluorescence microscope with a 60× objective (Olympus Fluoview FV3000).
4
Oxidative Stress and Apoptosis Assays
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Chemicals were the highest grade available. Norepinephrine bitartrate salt, apocynin, propranolol, phentolamine, superoxide dismutase-polyethylene glycol (PEG-SOD), and catalase-polyethylene glycol (PEG-CAT) were purchased from Sigma Chemicals. Phospho-p47phox (Ser370) was purchased from Invitrogen. Caspase-3 inhibitor III, was purchased from Santa Cruz Biotechnologies (Dallas, TX). FITC Annexin V/Dead Cell Apoptosis Kit®, CellROX® probe, 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein, CellEventTM® kit, Alexa Fluor ™ 647 goat anti-rabbit antibody, and one step Power SYBR™ Green RNA to CT™ RT-PCR kit were purchased from Thermo Fisher Scientific (Waltham, MA). FragEL™ DNA Fragmentation Detection Kit Fluorescence TdT enzyme was purchased from EMD Millipore (Billerica, MA).
5
Antibody-based Signaling Pathway Analysis
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Phospho-CSF1R (Tyr723) (49C10) Rabbit mAb (RRID: AB_2085229), β-catenin (6B3) Rabbit mAb (RRID: AB_331149), anti-Phospho-Akt (Ser473) (RRID: AB_329825) and anti-total-Akt antibodies (RRID: AB_329827) were all bought from Cell Signaling Technology. Anti-GAPDH (RRID: AB_2630358), anti-CSF1R (RRID: AB_2085251), anti-Aβ (MOAB2), anti-β-actin antibodies (RRID: AB_306371) and Alexa Fluor 647 Donkey Anti-Rat IgG H&L (RRID: AB_2813835), as well as the BrdU Cell proliferation ELISA Kit, were all from Abcam. Rat Anti-mouse CD68 monoclonal antibody (RRID: AB_322219) was from Bio-Rad. Anti-HA-tag (RRID: AB_11042321) antibody and anti-Myc-tag antibody (RRID: AB_11182162) were from Proteintech. Rabbit anti-Iba1 (RRID: AB_2687911) was from Wako. Alexa Fluor 488 Goat anti-Rabbit (RRID: AB_10374301), Alexa Fluor 594 Goat anti-Rabbit (RRID: AB_10374440), Alexa Fluor 647 Goat anti-Rabbit antibodies (RRIDAB_10371940) and TRIzol reagent were all from Thermo Fisher Scientific. Granulocyte-macrophage colony stimulating factor (GM-CSF) and CSF1 were from R&D Systems. Basic Glial Cells Nucleofector Kit was purchased from LONZA. ReverTra Ace qPCR RT Master Mix was from TOYOBO. FastStart Universal SYBR Green Master mix was from Roche. CellTiter 96R Aqueous One Solution (MTS assay) and DeadEnd™ Fluorometric TUNNEL System were from Promega.
6
Immunofluorescent Staining of GCGR
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After the glucose, glucagon or IBMX treatment, cells were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Then cells were incubated with the blocking solution (3% BSA in PBS) for 30 min at room temperature. Staining was performed incubating samples with the primary GCGR antibodies (2 μg/mL in 3% BSA) overnight at 4°C, and washed with PBS for three times. At last, samples were incubated with Alexa Fluor 647 goat anti-rabbit antibodies (2 μg/mL in 3% BSA; Thermo Fisher Scientific, A-21245) for 30 min at room temperature. After three washes, 50 μL imaging buffer containing Tris (50 mM, pH 8.0), NaCl (10 mM), glucose (10% w/v), glucose oxidase (500 μg/mL; Sigma), catalase (40 μg/mL; Sigma) and β-mercaptoethanol (1% v/v; Sigma) was dropped on a large microscope slide (24 mm × 50 mm, Fisher), and the small slide (22 mm × 22 mm, Fisher) where cells were seeded was covered on the large one and sealed with nail polish.
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