Cryosections were postfixed in 4% paraformaldehyde for 15 min, blocked with 2% BSA/TBST for 30 min and incubated with rabbit anti-GFP polyclonal antibody (1:200, Abcam ab290) or rabbit anti-periostin polyclonal antibody (1:2,000, Millipore ABT280) overnight at 4 °C, and subsequently with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen A11034) or Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen A11035) for 3 h at 4 °C. Sections were further incubated with DAPI (4′,6-diamidino-2-phenylindole) to stain nuclei. To immunohistochemically detect PPR, freshly isolated P7 mandibles were embedded in super cryosection embedding medium (Section-Lab, Hiroshima, Japan) on dry ice. Sections of mandibles were cut without decalcification using a tungsten blade and a tape transfer system (Cryofilm IIC; Section-Lab). Non-decalcified sections were adhered on the adhesive side of the cryofilm and analyzed as attached throughout. Sections on cryofilms were dried completely, postfixed in 4% paraformaldehyde for 15 min, blocked with 3% BSA/TBST for 30 min and incubated with rabbit anti-PPR polyclonal antibody (1:200, AssaybioTech G220) overnight at 4 °C, and subsequently with Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen A11035) for 3 h at 4 °C. Sections were further incubated with DAPI to stain nuclei.
Alexa fluor 546 conjugated goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
Alexa Fluor 546-conjugated goat anti-rabbit IgG is a secondary antibody used for immunofluorescence detection. It is a goat-derived antibody that specifically binds to rabbit immunoglobulin G (IgG) and is conjugated to the Alexa Fluor 546 fluorescent dye.
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Lab products found in correlation
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (14 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Wiseven woc high clean air oven, by Daihan Scientific (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 546 conjugated goat anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 conjugated donkey anti goat igg, by Thermo Fisher Scientific (3 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (2 mentions)
Slowfade antifade kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Canada balsam, by Kanto Chemical (2 mentions)
Axio observer z1, by Zeiss (2 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 633 conjugated goat anti guinea pig igg, by Thermo Fisher Scientific (2 mentions)
Cellsens dimension software, by Olympus (2 mentions)
Ma5 17040, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated rabbit anti rat igg, by Merck Group (2 mentions)
Cy3 conjugated donkey anti sheep igg, by Jackson ImmunoResearch (2 mentions)
Lsm 510 confocal microscope, by Zeiss (2 mentions)
52 protocols using alexa fluor 546 conjugated goat anti rabbit igg
1
Immunofluorescence Staining of Cryosections
2
Visualizing DMPK Kinase Activity in MCF-7 Cells
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MCF-7 cells were transfected with the expression plasmid for HA-DMPK-wild-type (WT) or HA-DMPK-kinase dead mutant (MT). The cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and then blocked with 2% BSA in phosphate buffered saline (PBS), the cells were incubated with anti-HA and anti-pMLC2 antibodies. Alexa Fluor 546-conjugated goat anti-rabbit IgG (Molecular Probes, Carlsbad, CA, USA) and Alexa Fluor 647-conjugated goat anti-mouse IgG (Molecular Probes, Carlsbad, CA) were used as secondary antibodies. Alexa Fluor 488 Phalloidin and DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) were used to stain actin filaments and nuclei, respectively. Images were acquired using a confocal microscope (A1R HD25, Nikon) and then analyzed with ImageJ software (NIH).
3
Immunofluorescence Staining of MMAB and SMAD4 in HeLa Cells
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HeLa cells were fixed in 4% paraformaldehyde on an eight-well cell culture slide (SPL Life Sciences) for double immunofluorescence staining. After fixation, cells were blocked in 5% non-fat milk for 60 min, followed by incubation with primary antibodies against MMAB and SMAD4 at 1:100 dilutions overnight at 4°C. Then, cells were incubated in the secondary antibodies, Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100; Molecular Probes, USA) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (1:100; Molecular Probes), for one hour at room temperature (RT). Finally, samples were washed in phosphate-buffered saline (PBS)/1% Triton X-100 and mounted. Cell nuclei were stained with Hoechst 33342 (Invitrogen). Slides were imaged using an LSM 700 ZEISS laser scanning confocal microscope (Carl Zeiss).
4
Immunofluorescence Assay for Smad3 in Pancreatic Cancer Cells
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The PANC-1 cells were seeded on coverslips, which were placed in six-well plates in advance. The following day, cells were transfected with miR-891b mimic/NC or siCbl-b/NC. After incubation for 48 hours, the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, blocked with 1% bovine serum albumin for 1 hour and incubated with anti-Smad3 antibody at 4°C overnight. The following day, the cells were rinsed with PBS and incubated with Alexa Fluor 546-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) for 1 hour at room temperature in the dark. 4′, 6-Diamidino-2-phenylindole (Sigma-Aldrich) was used to stain nuclei for 5 minutes. Following mounting with the antifade mounting medium (Beyotime Institute of Biotechnology, Haimen, China), the cells were visualized by fluorescence microscopy (BX60, Olympus, Tokyo, Japan).
5
Antibody Detection in Medaka Fish
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Primary antibodies were: (1) FXYD12: a rabbit polyclonal antibody (LTK BioLaboratories, Taoyuan, Taiwan) targeting the C-terminus of medaka FXYD12 (S2 Fig ); (2) NKA: a mouse monoclonal antibody (α5; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) against the α-subunit of avian NKA; and (3) actin: a rabbit polyclonal antibody (sc-1616-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA) against the C-terminus of human actin, as a loading control for immunoblotting. The specificity and related information for these antibodies have been demonstrated and described previously [25 (link), 29 ].
The secondary antibodies employed for immunofluorescent staining were Alexa-Fluor-546-conjugated goat anti-rabbit IgG and Alexa-Fluor-488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA). For immunoblotting, the secondary antibodies were horseradish-peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (Pierce, Rockford, IL, USA).
The secondary antibodies employed for immunofluorescent staining were Alexa-Fluor-546-conjugated goat anti-rabbit IgG and Alexa-Fluor-488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA). For immunoblotting, the secondary antibodies were horseradish-peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (Pierce, Rockford, IL, USA).
6
Immunofluorescence Analysis of Epithelial-Mesenchymal Transition
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The cells were seeded in Lab-Tek chamber slides (Nunc S/A, Polylabo, Strasbourg, France). After starved overnight, the cells were treated with or without IGF-I (100 ng/mL) for 48 hours and fixed in 3.3% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% bovine serum albumin (BSA) for 1 hour and then incubated with anti-E-cadherin and anti-Vimentin antibody overnight at 4°C. The next day, Alexa Fluor 546-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) were added in blocking solution for 1 hour at room temperature in the dark. 4′6′-diamidino-2- phenylindole was used to stain nuclei for 5 min. After mounted with the Slow Fade Antifade Kit (Molecular Probes, Eugene, OR, USA), the cells were visualized by fluorescence microscopy (BX61, Olympus, Japan).
7
Collagen VI Immunofluorescence in Skin and Wounds
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Immunofluorescence microscopy was performed on frozen sections (7 µm) of healthy or fibrotic skin and of wounds from wild type and Col6a1 null mice [32] (link). Sections were fixed with 2% paraformaldehyde and treated with bovine testicular hyaluronidase. Primary antibodies were applied over night at 4°C followed by incubation with secondary AlexaFluor 546-conjugated goat anti-rabbit IgG (Molecular Probes), AlexaFluor 488-conjugated goat anti-rabbit IgG (Molecular Probes), AlexaFluor 546-conjugated goat anti-guinea pig IgG, or AlexaFluor 546-conjugated rat anti-mouse pig IgG (Molecular Probes). The antibodies raised against recombinant N-terminal fragments of the collagen VI α3, α5 and α6 chains have been described [15] (link) and those against the collagen VI α1 and α2 chains were raised by immunization with recombinant C-terminal fragments of these chains. The ER marker protein disulfide isomerase (PDI) was detected with a purified anti-rabbit PDI antibody (Stressgene), the endothelial marker CD31 with a purified rat anti-mouse CD31 antibody (MEC13.3, BD-Pharmingen) and nerves with a purified rat anti-mouse neurofilament antibody (Dako). Nuclei were stained with DAPI (Sigma Aldrich).
8
Immunostaining of Skin Cross-Sections
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The immunostaining of skin cross-sections was performed as described26 (link). The cross-sections (8-μm thickness) were incubated with rat monoclonal anti-CD68 antibody (1:200; Abcam, Cambridge, MA), rat monoclonal anti-CXCR4 (1:200; R&D Systems, Minneapolis, MN), goat polyclonal anti-PDGFRα (1:20; R&D), or rabbit polyclonal anti-SDF-1α (1:200; eBioscience) at 4 °C overnight. The TECs were fixed in 4% paraformaldehyde (PFA) for 15 min, then washed and immunostained with rat monoclonal anti-CXCR4 (1:200; R&D) and goat polyclonal anti-PDGFRα (1:20; R&D) at 4 °C overnight. These cells were then stained with a secondary antibody, Alexa Fluor 546-conjugated goat anti-rabbit IgG (1:400; Molecular Probes, Eugene, OR). Stained cross-sections and cells were visualised using a confocal laser microscope (model A1/C1; Nikon, Tokyo) using NIS-Elements AR 3.1 software, and the images were arranged with Adobe Photoshop software.
9
Inhibition of Phospholipase C Signaling
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The PLC inhibitor U73122 and its inactive analogue U73343 were purchased from Calbiochem (San Diego, CA). Antibodies against rabbit anti-phospho-TrkA (Tyr490) (catalog #9141), anti-phospho-PLCγ-1 (Ser1248) (catalog #4510), anti-phospho-PLCγ-2 (Tyr759) (catalog #3874), anti-phospho-PLCβ-3 (Ser537) (catalog #2481), anti- PLCγ-1 (catalog #2822), and anti-β-actin (catalog #4967) were obtained from Cell Signaling Technology (Danvers, MA). BODIPY FL C5-ganglioside GM1a and Alexa Fluor 546-conjugated goat anti-rabbit IgG (catalog #A-11010) were purchased from Molecular Probes (Eugene, OR). Horseradish peroxidase-labeled goat anti-rabbit IgG (catalog #7074) and an ECL Western blotting kit were obtained from GE Healthcare (Tokyo, Japan). Hoechst 33342 was obtained from Dojindo Laboratories (Kumamoto, Japan). All other drugs were of analytical grade.
10
Immunofluorescence Analysis of EMT Markers
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The cells were seeded in Lab-Tek chamber slides (Nunc S/A, Polylabo, Strasbourg, France). The cells were treated with or without TAM (5 μmol/L) for 72 h and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% bovine serum albumin (BSA) in 1× PBS for 1 h at room temperature and then incubated with E-cadherin and vimentin antibodies for 1 h. Then Alexa Fluor 546-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) were added in blocking solution for 1 h at room temperature in the dark. 4′6-diamidino-2- phenylindole was used to stain nuclei for 5 min. After mounted with the Slow Fade Antifade Kit (Molecular Probes, Eugene, OR, USA), the cells were visualized by fluorescence microscopy (BX61, Olympus, Japan) [16 (link)].
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