Colon sections were prepared as described above, deparaffinized, and rehydrated in descending ethanol series before heat-induced antigen retrieval (Vector Lab, Burlingame, CA), blocked for 3 hours in PBS containing 10% goat or horse serum (MP Biomedicals, Solon, OH), and then incubated overnight with primary antibodies against either LYVE-1 (1:150; Abcam), macrophage galactose-specific lectin-2 (Mac-2, 1:10,000; Accurate Chemical, Westbury, NY), VEGF-C (1:100; Santa Cruz), or VEGF-D (1:100; Santa Cruz). Slides were rinsed in TBS-T after incubation with Alexa Fluor 546-conjugated goat anti-rat or Alexa Fluor 647-conjugated goat anti-rabbit, Alexa Fluor 647-conjugated donkey anti-goat, Alexa Fluor 546-conjugated donkey anti-rat, or Alexa Fluor 488-conjugated donkey antirabbit immunoglobulin G (1:200; ThermoFisher Scientific) antibodies for 2 hours at RT. Slides were costained with 4,6-diamidino-2-phenylindole (DAPI; ThermoFisher Scientific) and mounted with N-propyl gallate (Sigma).
Alexa fluor 647 conjugated goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647-conjugated goat anti-rabbit is a secondary antibody that binds to rabbit primary antibodies. The Alexa Fluor 647 dye is used for fluorescent detection and visualization.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 488 conjugated α bungarotoxin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igm, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated goat anti mouse igg1, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Laminin, by Merck Group (2 mentions)
Alexa fluor 647 conjugated goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Paraformaldehyde (pfa), by AppliChem (2 mentions)
Poly d lysine covered chamber slides, by Corning (2 mentions)
Moviol, by Merck Group (2 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Bouin s fixative, by Polysciences (2 mentions)
Slowfade diamond antifade mountant containing dapi, by Thermo Fisher Scientific (2 mentions)
A 21245, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti chicken, by Thermo Fisher Scientific (2 mentions)
29 protocols using alexa fluor 647 conjugated goat anti rabbit
1
Immunohistochemical Evaluation of Lymphatic Markers
2
Immunofluorescent Staining of Cultured Fibroblasts
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Cultured and treated primary capsular fibroblasts were fixed using 100% methanol fix for 15 minutes. After fixing, cells were rinsed in 1× PBS. Cells were then blocked using 5% BSA in 1× PBS for 1 hour with gentle rocking at room temperature. Cells were incubated with primary αSMA antibody (rabbit, 1:1,000, Abcam, Cambridge, UK) for 1 hour at room temperature (RT). After primary incubation, antibody solution was removed and washed with 1× PBS 3 times. Cells were then incubated with secondary antibody (AlexaFluor 647 conjugated goat antirabbit, 1:500, ThermoScientific, Waltham, MA) for 45 minutes at RT. The secondary solution was removed and then washed 3 times with 1× PBS before visualizing with an EVOS cell imaging system (Invitrogen, Carlsbad, CA).
3
Immunofluorescence Analysis of NLRC5 Expression
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Cells were seeded at 1 × 105 cells/well on glass-bottomed culture dishes and then fixed with freshly prepared 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min. Human artery and mouse common carotid artery tissues were harvested and processed in optimal cutting temperature compound and sliced into 5 μm-thick sections. Paraformaldehyde-fixed sections, cryosections, and cells were incubated with anti-NLRC5 (ab105411, Abcam), anti-α-smooth muscle actin (α-SMA, ab7817, Abcam, 1:200), anti-PPARγ (sc-7196, Santa Cruz, 1:100), anti-CD31 (557355, BD Bioscience, USA, 1:100), anti-FLAG (ab49763, Santa Cruz, 1:100), and anti-myc (2276, Cell Signaling Technology, 1:100) overnight at 4 °C. Normal isotype IgG (sc2027, Santa Cruz) was used as negative control. After washing with phosphate-buffered saline (PBS), secondary antibodies (Alexa Fluor 647-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-mouse; Thermo Fisher Scientific, 1:200) were incubated for 1 h at 37 °C in the dark. Nuclei were labeled with DAPI (Vector Laboratories), and cells were visualized using an LSM710 laser confocal microscope (Carl Zeiss, Germany).
4
Double Immunostaining of hHSPB1 in Brain Sections
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Double immunostaining was performed to study the expression pattern of hHSPB1. After washing in PBS, 30-μm-thick frozen sagittal brain sections were permeabilized and blocked with 0.2% Triton X-100 and 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Then the sections were incubated overnight at 4 °C with the following primary antibodies: goat anti-IBA1 (ionized calcium–binding adaptor molecule 1, Abcam, Cambridge, UK), mouse anti-GFAP (Sigma-Aldrich Ltd, Budapest, Hungary), mouse anti-NEUN (neuronal nuclei, Merck Millipore, MA, USA), and rabbit anti-hHSPB1. Appropriate secondary antibodies were applied for 2 h: Alexa Fluor-488–conjugated rabbit anti-goat (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK); FITC-conjugated goat anti-mouse (Sigma-Aldrich Ltd, Budapest, Hungary); Alexa Fluor-647–conjugated goat anti-rabbit (Thermo Fisher Scientific, MA, USA) (Table S1 ). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Ltd, Budapest, Hungary), at the concentration of 0.5 μg/ml for 5 min. Immunostainings were examined with a confocal laser scanning microscope (Olympus Fluoview FV1000, Olympus Life Science Europa GmbH, Hamburg, Germany).
5
Immunohistochemical Analysis of Muscle Fiber Types
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Pax7 (mouse IgG1, 1:100, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa), Laminin (rabbit, 1:1500, Sigma-Aldrich L9393), GFP (rabbit, 1:400, Millipore AB3030P, Billerica, Massachusetts), SC-71 (MyHC-IIA, mouse IgG1, 1:40, DSHB), BF-F3 (MyHC-IIB, mouse IgG1, 1:40, DSHB), SV2 (mouse IgG1, 1:100, DSHB), Znp-1 (Syt-2, mouse IgG1, 1:200, DSHB) and 2H3 (neurofilament, mouse IgG1, 1:200, DSHB), AlexaFluor 488-conjugated α-Bungarotoxin (1:1000, Thermo Fisher Scientific B-13422), AlexaFluor 647-conjugated α-Bungarotoxin (1:1000, Thermo Fisher Scientific B-35450), AlexaFluor 594-conjugated goat anti-mouse IgG (1:1500, Thermo Fisher Scientific A-11032), AlexaFluor 594-conjugated goat anti-mouse IgG1 (1:1500, Thermo Fisher Scientific A-21125), AlexaFluor 488-conjugated goat anti-mouse IgM (1:1500, Thermo Fisher Scientific A-21042), AlexaFluor 488-conjugated goat anti-rabbit IgG (1:1500, Thermo Fisher Scientific A-32731), AlexaFluor 647-conjugated goat anti-rabbit (1:1500, Thermo Fisher Scientific A-21244).
6
Immunofluorescent Staining of DENV Proteins
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Cells were grown on glass coverslips, fixed in 4% (w/v) paraformaldehyde at room temperature for 10 min and quenched with 50 mM NH4Cl for 5 min. Cells were then permeabilized with 0.1% (v/v) Triton X-100 for 15 min and blocked in 10% (v/v) normal goat serum (Sigma Aldrich) for 15 min. DENV-2 NS3 protein was detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich). DENV-2 E and C were probed with a rabbit anti-DENV E polyclonal antibody (GeneTex) and a rabbit anti-DENV C polyclonal antibody (Novusbio), respectively. Double strand (ds) RNA was probed with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS). The secondary antibodies were Alexa Fluor 647-conjugated goat anti-rabbit (Thermo Fisher Scientific) or Cy5-conjugated goat anti-mouse (Life Technologies). Nuclei were stained with 1 µM 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). The coverslips were mounted onto slides with ProLong Gold antifade reagent (Life Technologies). Fluorescent images were captured using a Zeiss 780 NLO confocal scanning microscope (Zeiss) with 63× objective lenses and standard lasers and filters for Alexa Fluor 647, Cy5, and DAPI fluorescence.
7
Immunolabeling of Neuromuscular Junctions
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Pax7 (mouse IgG1, 1:100, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA), BrdU (rat, 1:250, Abcam ab6326, Cambridge, UK), Laminin (rat or rabbit, 1:1500, Sigma–Aldrich L0663 or L9393), GFP (rabbit, 1:400, Millipore AB3030P, Billerica, MA), F1.652 (mouse IgG1, 1:40, DSHB), A4.840 (mouse IgM, 1:40, DSHB), NCL-MHC (mouse IgG1, 1:100, DSHB), SC-71 (mouse IgG1, 1:40, DSHB), 6H1 (mouse IgM, 1:40, DSHB), BF-35 (mouse IgG1, 1:40, DSHB), BF-F3 (mouse IgG1, 1:40, DSHB), SV2 (synaptic vesicle protein-2, mouse IgG1, 1:100, DSHB), Znp-1 (synaptotagmin-2, mouse IgG1, 1:200, DSHB) and 2H3 (neurofilament, mouse IgG1, 1:200, DSHB), AlexaFluor 488-conjugated α-Bungarotoxin (1:1000, Life Technologies B-13422, Grand Island, NY), AlexaFluor 647-conjugated α-Bungarotoxin (1:1000, Life Technologies B-35450), AlexaFluor 594-conjugated goat anti-mouse IgG (1:1500, Life Technologies A-11032), AlexaFluor 594-conjugated goat anti-mouse IgG1 (1:1500, Life Technologies A-21125), AlexaFluor 488-conjugated goat anti-mouse IgM (1:1500, Life Technologies A-21042), AlexaFluor 488-conjugated goat anti-rat IgG (1:1500, Life Technologies A-11006), AlexaFluor 647-conjugated goat anti-rabbit (1:1500, Life Technologies A-21244).
8
Immunodetection of Membrane Proteins
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Primary antibodies included rabbit anti-MUC1 C-terminus CT-1 [49 (link)], Armenian hamster anti-MUC1 C-terminus CT-2 (MH1, ProSci Inc., Poway, CA), mouse anti-EGFR (H9B4, Thermo Scientific), mouse anti-EGFR (H11, Thermo Scientific), mouse anti-β-actin (ab8226, Abcam, Cambridge, UK), rabbit anti-EGFR-phospho-Y1068 (Invitrogen), rabbit anti-ERK (Cell Signaling Technology, Danvers, MA), rabbit anti-phospho-ERK (E10, Cell Signaling Technology), and rabbit anti-Ki67 (Novus Biologicals Inc., Littleton, CO). Secondary antibodies for western blotting included horseradish peroxidase-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO). Secondary antibodies for immunofluorescence included AlexaFluor-488-conjugated goat anti-mouse (Life Technologies, Carlsbad, CA), AlexaFluor-488-conjugated goat anti-rabbit (Life Technologies), AlexaFluor-568-conjugated goat anti-rabbit (Life Technologies), AlexaFluor-647-conjugated goat anti-Armenian hamster (Fisher Scientific), AlexaFluor-647-conjugated goat anti-rabbit (Life Technologies) and AlexaFluor-647-conjugated goat anti-mouse (Life Technologies).
9
Autofluorescence and Immunofluorescence Analysis of Cells
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For the microscopic analysis of autofluorescence, cells were grown on coverslips for 16hr, treated with 50 μM for 3 h before being fixed with 4% PFA. After mounting with Fluoromont-G mounting medium (Southern biotech), coverslips were observed with a Zeiss confocal microscope and fluorescence spectra induced by menadione treatment was evaluated in multiple regions of interest (ROI) within the same cell.
For indirect immunofluorescence analysis, cells were fixed on coverslips for 15 min with 4% PFA and then permeabilized for 5 min with Triton 0.2% at room temperature. AIF protein was detected by incubation with an anti-AIF pAB (1:100 dilution in PBS / 1% BSA / 2% goat serum) followed by Alexafluor-647-conjugated goat anti-rabbit (Life Technologies). DNA was stained with Hoechst 3342 or TO-PRO-3 (Life Technologies). Sample slides were then mounted using the reagent Fluoromont-G (Southern Biotech) and observed by confocal microscopy (LSM-510; Carl Zeiss Microimaging) equipped with an X63 objective.
For indirect immunofluorescence analysis, cells were fixed on coverslips for 15 min with 4% PFA and then permeabilized for 5 min with Triton 0.2% at room temperature. AIF protein was detected by incubation with an anti-AIF pAB (1:100 dilution in PBS / 1% BSA / 2% goat serum) followed by Alexafluor-647-conjugated goat anti-rabbit (Life Technologies). DNA was stained with Hoechst 3342 or TO-PRO-3 (Life Technologies). Sample slides were then mounted using the reagent Fluoromont-G (Southern Biotech) and observed by confocal microscopy (LSM-510; Carl Zeiss Microimaging) equipped with an X63 objective.
10
Immunostaining of Cultured Cells
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Cells were cultured overnight on poly-D lysine-covered chamber slides (Corning), washed with TBS, fixed in 4% paraformaldehyde (AppliChem) for 15 minutes, and permeabilized with 0.1% Triton-X in TBS. After blocking in 3% (w/v) BSA (Sigma) and 0.1% Tween20 in TBS for 1h cells were incubated with primary Abs. After extensive washing in TBS-T, we stained the cells with Alexa Fluor 647-conjugated goat anti-rabbit, Alexa Fluor 488-conjugated goat anti-mouse, or Alexa Fluor 647-conjugated mouse anti-rat secondary Abs (Life Technologies). Nuclei were stained with DAPI (Life Technologies). The slides were mounted with Moviol (Sigma). Images were acquired by a Leica TCS-SP5 confocal microscope (Leica Microsystems) under identical conditions for pinhole opening, laser power, photomultiplier tension, and layer number. During data elaboration by Imaris (Switzerland), identical parameters were applied to all samples.
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