NH4Cl, 2,2′-dipyridyl, CoCl2, Dulbecco’s modified Eagle’s medium (DMEM), ScreenFect™ A, an anti-DYKDDDDK (FLAG) antibody, and an anti-Myc tag monoclonal antibody were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Penicillin–streptomycin solution, fetal bovine serum (FBS), geneticin (G418), CHX, bafilomycin A1, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). MG132 (Z-Leu-Leu-Leu-CHO) was purchased from the Peptide Institute (Osaka, Japan). Isogen was from Nippon Gene (Toyama, Japan), Revert Aid™ M-MuLV Reverse Transcriptase from MBI Fermentas (Vilnius, Lithuania), Go Taq polymerase from Promega (Madison, WI), and KOD Fx Neo from Toyobo (Tokyo, Japan). The DNA Ligation Kit was obtained from Takara Bio Inc. (Shiga, Japan). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Dojindo (Kumamoto, Japan). Anti-Parkin and anti-Hsc70 antibodies were from ProteinTech (Rosemont, IL, USA). An anti-ubiquitin antibody (clone FK2) was from StressMarq Bioscience. Alexa Fluor® 594-conjugated goat anti-mouse IgG and Alexa Fluor® 647-conjugated goat anti-rabbit IgG were obtained from Abcam (Carlsbad, CA, USA).
Alexa fluor 647 conjugated goat anti rabbit igg
Manufactured by Abcam
Sourced in United States
Alexa Fluor 647-conjugated goat anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 647 fluorescent dye. It is designed to specifically bind to and detect rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid m mulv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (2 mentions)
Penicillin streptomycin solution, by Merck Group (2 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Abcam (2 mentions)
Anti dykddddk flag antibody, by Fujifilm (2 mentions)
Anti myc tag monoclonal antibody, by Fujifilm (2 mentions)
Isogen, by Nippon Gene (2 mentions)
Eclipse ti2 series, by Nikon (2 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Dna ligation kit, by Takara Bio (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Screenfect a, by Fujifilm (1 mentions)
Gotaq polymerase, by Promega (1 mentions)
Nh4cl, by Fujifilm (1 mentions)
2 2 dipyridyl, by Fujifilm (1 mentions)
Cocl2, by Fujifilm (1 mentions)
Mg132 z leu leu leu cho, by Peptide Institute (1 mentions)
Dulbecco s modified eagle s medium dmem, by Fujifilm (1 mentions)
Kod fx neo, by Toyobo (1 mentions)
Cytokeratin 18, by Abcam (1 mentions)
13 protocols using alexa fluor 647 conjugated goat anti rabbit igg
1
Analyzing Parkin-Mediated Protein Degradation
2
Lipofuscin Characterization in Liver Cells
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The presence of lipofuscin, a highly oxidized insoluble protein that accumulates in the cytoplasm, was identified using Sudan Black B (SBB) and Fontana Masson staining only at 6 months of age. Pictures were quantified using ImageJ software, and a “stack image” and a color threshold were applied to identify the stained structure. Results are reported as a red stained area percentage among the total area [30 (link),42 (link)]. Additionally, the autofluorescence of liver lipofuscin was detected using an inverted fluorescent microscope (Eclipse Ti2 Series-Nikon) at birth and 6 months of age. Lipofuscin exhibits broad-spectrum autofluorescence [43 (link),44 (link)]. A GFP filter set was applied with an exposure time of 400 ms throughout the observation. Pictures were taken by a single examiner (C.Y.).
To localize lipofuscin deposits at 6 months of age, hepatocytes were stained with cytokeratin 18 (1/100, Abcam) overnight at 4 °C. Sections were then washed with PBS and incubated for two hours with Alexa Fluor-647-conjugated goat anti-rabbit IgG (1/200, Abcam). Sections were then rinsed with PBS and mounted using Fluoromount g mounting medium with DAPI. A negative control was established through incubation only with secondary antibody. Slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y.).
To localize lipofuscin deposits at 6 months of age, hepatocytes were stained with cytokeratin 18 (1/100, Abcam) overnight at 4 °C. Sections were then washed with PBS and incubated for two hours with Alexa Fluor-647-conjugated goat anti-rabbit IgG (1/200, Abcam). Sections were then rinsed with PBS and mounted using Fluoromount g mounting medium with DAPI. A negative control was established through incubation only with secondary antibody. Slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y.).
3
Comprehensive Biomaterial Characterization for Cell Studies
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CS was purchased from Aladdin biochemical technology Co., Ltd. (Shanghai, China); Doxorubicin hydrochloride was purchased from Meilun Biotechnology (Dalian, China); BSA (Albumin Bovine V) was purchased from BioFroxx (Einhausen, Germany); Soybean oil for injection was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China); Solutol HS15 was purchased from BASF (Ludwigshafen, Germany); 3-(4,5-Dimethyl-2-thiazolyl)−2,5-diphenyl-2H-tetrazolium bromide (MTT), 4,6-diamidino-2-phenylindole (DAPI), monensin sodium and genistein were purchased from J&K Scientific Ltd.; Mouse antibodies including anti-CD44, FITC anti-CD44 and Rhodamine-labeled secondary antibodies were purchased from Affymetrix eBioscience (San Diego, USA). rabbit anti-CD31 antibody and Alexa Fluor 647 conjugated goat anti-rabbit IgG were purchased from Abcam (Cambridge, UK). Other reagents were analytical or HPLC grade and purchased commercially.
4
Immunofluorescence Assay for γH2AX
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HepG2 and Hep3B cells were plated onto glass coverslips coated with gelatin in 24-well plates. Then, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton-X 100 for 15 min, and blocked with 5% BSA in PBS for 30 min. Subsequently, cells were incubated with anti-γH2AX antibody (1:1000 dilution, ab229914, abcam, USA) for 1.5 h, followed by incubation with Alexa Fluor® 647 conjugated goat anti-rabbit IgG (1:1000 dilution, ab150079, abcam, USA) for 1 h. Nuclear was counterstained with 1 µg/mL DAPI solution (Thermo Fisher Scientific, USA). Stained cells were visualized with a Zeiss LSM700 confocal microscope (Carl Zeiss, Germany).
5
Nrf2 Regulation and Cellular Homeostasis
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Dulbecco's modified Eagle's medium, tBHQ, hydrogen peroxide, an anti-DYKDDDDK (FLAG) antibody, anti-Myc tag monoclonal antibody, anti-GFP(VC) antibody (mFX75), and horseradish-peroxidase-conjugated goat anti-mouse IgG were purchased from Wako Pure Chemical Industries. MG132 (Z-Leu-Leu-Leu-H) was purchased from the Peptide Institute. Mithramycin A was from Cayman Chemicals. Penicillin-streptomycin solution, fetal bovine serum, and geneticin (G418) were from Sigma Chemical Co. Nitrocellulose membrane, horseradish-peroxidase-conjugated goat anti-rabbit IgG, and 4-chloro-1-naphthol were purchased from Bio-Rad Laboratories. Isogen was from Nippon Gene, and Revert Aid M-MuLV Reverse Transcriptase was from MBI Fermentas. KOD Plus Neo and Fx Neo DNA polymerase were from Toyobo. DAPI (4′,6-diamidino-2-phenylindole) was from Dojindo. Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG were from Abcam. Anti-CUL4, anti-KLF15, and anti-AP2α antibodies were from Santa Cruz Biotechnology. An anti-Sp1 antibody was purchased from Cell Signaling Technology. An anti-ubiquitin antibody (clone FK2) was from StressMarq Bioscience. The anti-Nrf2, anti-Keap1, anti-WDR23, and anti-β-actin antibodies were prepared as described previously (12 (link), 55 (link)).
6
Apoptotic Neuron Identification via TUNEL and NeuN
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Double staining of TUNEL and the neuronal marker NeuN was performed to discern apoptotic neurons. Briefly, the spinal cord sections were stained with 2 μg/ml anti-NeuN antibody (Abcam ab128886) and then the Alexa Fluor 647-conjugated goat anti-rabbit IgG (Abcam ab150083). The sections were then subjected to staining with the TUNEL Assay Kit-BrdU-Red (Abcam ab66110) following the manufacturer’s manual. The percentage of TUNEL+NeuN+ cells in NeuN+ cells was calculated.
7
Quantification of His-tagged and Vimentin-positive Cells
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HSFs (5 × 106 cells) were harvested in clean EP tubes and stained with rabbit anti-His antibody (1:300, Abcam, Cambridge, USA) or rabbit anti-Vimentin antibody (1:300, Abcam, Cambridge, USA) at 4°C for 30 min. The stained HSFs were washed twice with 1 × PBS and then treated with Alexa Fluor 647-conjugated goat anti-rabbit IgG (1:2000, Abcam, Cambridge, USA) at 4°C for 1 h with light protection. The labeled HSFs were subjected to flow cytometry analysis using FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For cells that needed to block the vimentin receptors, HSFs were incubated with mouse anti-vimentin antisera (1:10) for 2 h after collection, washed twice with 1 × PBS, and performed the above experiments. The percentage of His-positive or vimentin-positive cells was determined using the FlowJo software package.
8
Quantifying 53BP1 in Liver Sections
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Liver sections from CTRL and IUGR males at birth and 6-month-old rats were stained with 53BP-1 (1/100, Abcam, Cambridge, UK) overnight at 4 °C. Sections were then washed with PBS and incubated for two hours with Alexa Fluor-647-conjugated goat anti-rabbit IgG (1/200, Abcam). Sections were then rinsed with PBS and mounted using Fluoromount g mounting medium with DAPI. A negative control was established through incubating only with secondary antibody. Slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y.) [30 (link)]. Fluorescence was evaluated with ImageJ software, and liver autofluorescence was subtracted
9
Immunocytochemical Analysis of α-SMA and Palladin
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The cells growing on a glass-bottomed culture dish (MatTek, Ashland, MA, United States) were fixed with cold acetone. Cells were then incubated with Alexa Fluor 488-conjugated rabbit IgG targeting alpha-smooth muscle actin (α-SMA) (2 μg/ml; Abcam). Rabbit anti-palladin IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG were used to detect palladin expression (2 μg/ml; Abcam). After 4′,6-diamidino-2-phenylindole incubation, the cells were observed under a digital confocal microscope (Leica, Wetzlar, Germany).
10
Immunofluorescence Analysis of Xenograft Tumors
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HCT116 xenograft tumour tissues were formalin‐fixed and paraffin‐embedded, then tissue slides were treated dewaxed and rehydrated slices to recover antigen using sodium citrate buffer. Cells with the indicated treatment were seeded on poly‐L‐lysine and collagen I‐coated cover glasses were fixed with 4% paraformaldehyde and preimmobilised with Triton X‐100 0.3%. After incubation with 10% normal goat serum for 1 h, the slices were incubated with anti‐TUNEL antibody or anti‐PCNA antibody (Abcam, UK) at 4°C overnight. Then the slices were washed and incubated with Alexa Fluor 647 conjugated goat anti‐rabbit IgG (Abcam) for 1 h. All nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole.
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