Slides were pretreated as described above for IHC. After blocking with 10% fetal bovine serum (GeneDirex, Anaheim, CA, USA) for 1 h, sections were incubated with a rabbit monoclonal antibody against human CD8 (clone SP16 Thermo Fisher, Waltham, MA, USA) for 45 min at 25°C temperature. Tissue sections were washed three times with TBS-Tween 20 at 60 rpm on a shaker for 5 min each and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Thermo Fisher, Waltham, MA, USA) for 45 min at 25°C followed by three PBS-Tween 20 washes at 60 rpm on a shaker for 5 min each. PD-1 staining was performed similarly except that a mouse monoclonal mouse antibody against human PD-1 (clone NAT105, Abcam, Cambridge, United Kingdom) at 1:100 dilution and an Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (Thermo Fisher, Waltham, MA, USA) at 1:500 dilution were used. Finally, sections were processed using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher, Waltham, MA, USA) for DAPI nuclear stain and mounting. IF images were captured using a Leica TCS SP8 X white light laser confocal microscope.
Alexa fluor 647 conjugated goat anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 647-conjugated goat anti-mouse secondary antibody is a fluorescently labeled antibody used to detect and visualize mouse primary antibodies in various immunoassays and imaging applications. The Alexa Fluor 647 dye provides a bright, photostable signal for sensitive detection.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by GeneDireX (1 mentions)
Tcs sp8 x white light laser confocal microscope, by Leica camera (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Pimozide, by Merck Group (1 mentions)
Poly l lysine coated coverslip, by Merck Group (1 mentions)
Evos fl microscope, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Alexa fluor 488 conjugated goat anti rat antibody, by Thermo Fisher Scientific (1 mentions)
Rad23a, by Cell Signaling Technology (1 mentions)
Clone da1e, by Cell Signaling Technology (1 mentions)
Clone 1b1b2, by Cell Signaling Technology (1 mentions)
Clone 13e5, by Cell Signaling Technology (1 mentions)
Clone m2, by Merck Group (1 mentions)
Clone m01, by Abnova (1 mentions)
Clone c29f4, by Cell Signaling Technology (1 mentions)
Flag antibody, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Paraformaldehyde solution, by Thermo Fisher Scientific (1 mentions)
10 protocols using alexa fluor 647 conjugated goat anti mouse secondary antibody
1
Multiplex Immunofluorescence Staining for CD8 and PD-1
2
Pimozide Treatment and Immunofluorescence Imaging
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Briefly, 1.5 × 105 cells were seeded on Poly-L-Lysine coated coverslips (Cat#P4707, Sigma-Aldrich) in 6 well plates (Cat#3516, Corning). Subsequently, cells were treated with vehicle (dimethyl sulfoxide (DMSO)) (Cat#D128-500, ThermoFisher Scientific) or 10 μM pimozide (Cat#P1793, Sigma-Aldrich) for 24 h. Cells were then fixed in 4% paraformaldehyde (Cat#159-SP, Electron Microscopy Sciences) for 15 min at room temperature. Fixed cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) (1x) and then permeabilized in 100% methanol for 10 min at −20°C. Following another DPBS (1x) wash, cells were blocked in blocking buffer (DPBS 1x, 5% normal serum, 0.3% Triton X-100) for 60 min at room temperature. Cells were then incubated with a given primary antibody (diluted 1:100 in DPBS 1x, 1% BSA, 0.3% Triton X-100) for 1 h at 37°C. After three DPBS (1x) washes, cells were incubated with an Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (diluted 1:500 in DPBS 1x, 1% BSA, 0.3% Triton X-100) for 1 h at 37°C (Cat#A-21236, ThermoFisher Scientific). Cells were washed three times with DPBS (1x) before being mounted with ProLong Gold Antifade with 4ʹ6-diamidino-2-phenylindole (DAPI) (Cat#P36931, ThermoFisher Scientific). Using the EVOS FL microscope, images were acquired at 40x objective using the Cy5.5 light cube (ThermoFisher Scientific).
3
Replication Fork Dynamics Visualization
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Cells were synchronized to the S phase by double thymidine block. Subsequently, CIdU (25 μM) was added for 20 min, followed by IdU (250 μM) for a further 40 min. Cells were harvested and washed twice with PBS. Cell samples of 2 μL (1,000 cells) were spotted onto the glass slide, air-dried for 5 min, and lysed in 7 μL lysis buffer (200 mM Tris-HCl, pH 7.5, 50 mM EDTA, 5% SDS) for 2 min. The slide was placed at an angle of 30 °C to allow the DNA to flow out slowly. Slides were air dried before fixation in methanol: acetic acid (3:1). Fixed fibers were denatured in 2.5 M HCl for 1 h before blocking in 5% BSA for 1 h and then incubated with anti-mouse BrdU antibody (BD Biosciences, 1:60), and anti-rabbit BrdU antibody (Sigma, 1:500) for 2 h, respectively. Slides were washed three times with 0.05% Tween-20 and PBS, followed by incubation with Alexa Fluor 488-conjugated goat anti-rat antibody (Thermo Fisher Scientific, 1:400) and Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, 1:400) for 1 h in the dark. Slides were washed three times with 0.05% Tween-20 and PBS. Slides were imaged using an Olympus microscope and the track lengths of the labeled replication forks were analyzed manually using Image J software.
4
Immunoprecipitation and Immunoblotting Protocol
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Immunoprecipitation and immunoblotting experiments were performed as previously described.34 (link) Antibodies against HA (1:2000; clone C29F4, cat # 3724S), β-actin (1:5000; clone 13E5, cat # 4970S), H3 (1:4000; clone 1B1B2, cat # 14269S) and RAD23A (1:1000; clone D7U7Z, cat # 24555) were purchased from Cell Signaling Technology, as was a non-specific isotype control IgG (clone DA1E, cat # 3900S). The FLAG antibody was from Sigma-Aldrich (1:4000; clone M2, cat # F1804). The Pol ι antibody was from Abnova (1:1000; clone M01, cat # H00011201-M01). Primary antibodies were detected with fluorescent secondary antibodies, and immunoblots quantified, as described previously.34 (link) For the microarray, the Pol ι antibody was detected with an Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific cat #A-21240).
5
Single-cell Immunostaining and Flow Cytometry
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Cell/MC aggregates were dissociated into single cells with TrypLE™ Express (Thermo Fisher Scientific, USA), and the dissociated cell/MC suspension was pipetted through a 40-μm nylon mesh Easystrainer® (Greiner Bio-One, Austria) to remove the MCs. Subsequently, the dissociated single cells were fixed in 4% paraformaldehyde solution in PBS (Fisher Scientific, USA) for 15 min at room temperature. Thereafter, the cells were incubated with primary antibodies (Supplementary Table 1 ) in 1% bovine serum albumin (BSA) (Sigma-Aldrich, USA) with 0.2% Triton X-100 (Sigma-Aldrich, USA) in PBS for 30 min. Then, the samples were washed with blocking buffer 1% BSA in PBS, followed by incubation for 20 min at room temperature in 1:500 dilution of Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, USA), Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific, USA) or Alexa Fluor 647 donkey anti-rabbit secondary antibody (Thermo Fisher Scientific, USA). Cells were washed with blocking buffer again and analysed on a flow cytometer (GUAVA easy Cyte 8HT, Millipore, USA) using standard filter sets for secondary antibodies. A minimum of 10,000 events were captured per sample. Death cell and cell debris were excluded. Analyses and gating were performed with Flowjo® 10.0.7 software (Tree Star Inc., USA).
6
Myosin Immunohistochemistry in Fish Tissue
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Myosin immunohistochemistry was performed as described [6 ]. Negative control experiments were performed in which either primary or secondary antibodies were omitted. Briefly, slides with coronal frozen sections (12 μm) were washed in PBS for 5 min and placed in blocking solution (5% goat serum in PBS + 0.2% Tween, PBST) for 30 min. Slides were incubated in a humidity chamber overnight at 4°C in primary antibody (mouse monoclonal anti-myosin heavy chain, F59, Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, Iowa) diluted to 1:50 in PBST+1% goat serum and washed again 4 times for 5 min in PBST. Then, slides were incubated in the dark with Alexafluor 647-conjugated goat anti-mouse secondary antibody (Invitrogen) diluted 1:1000 in PBST + 1% goat serum. After 3 5-min PBS washes, slides were coverslipped using ProLong Diamond Antifade Reagent with DAPI to stain the nuclei. Sections from 7 different fish per experiment were stained and imaged.
7
PAR-1 Receptor Cleavage Visualization
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HUVEC cells cultured on coverslips were stimulated with human MMP-9 for 5 minutes, as described above. The cells were then washed with HBSS without Ca2+ and Mg2+, fixed with 100% methanol, blocked with 5% BSA in PBS followed by incubation with anti-PAR-1 receptor antibody (ATAP-2, Zymed Laboratories Inc.; Bedminster, NJ). Anti-PAR-1 (ATAP-2) antibody recognizes intact and cleaved forms of PAR-1 receptor. Finally, cells were incubated with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Invitrogen; Waltham, MA). Cleavage of PAR-1 receptor was visualized using Perkin Elmer Ultra VIEW LCI confocal imaging system with Nikon TE2000-S fluorescence microscope and PlanApo360 immersion oil objective (numerical aperture 1.4) at room temperature. Ultra VIEW Imaging Suite software (version 5.5.0.4) was used for image processing.
8
Immunofluorescence Staining of HeLa Cells
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HeLa cells were seeded onto 20 mm diameter glass coverslips and transfected. The transfected cells were fixed with 4% paraformaldehyde (in PBS) for 15 min at room temperature, washed with PBS, and permeabilized with 0.5%Triton-X 100 in PBS for 10 min, then incubated in blocking buffer (5% BSA and 5% FBS in PBS) for 1 h. Cells were sequentially stained with mouse anti-importin-β antibody (Invitrogen, catalog number MA3-070, 1:500 dilution) or rabbit anti-FLAG antibody (Cell Signaling Technology, catalog number 14793, 1:1,000 dilution) for 2 h, and with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Invitrogen, catalog number A21237, 1:500 dilution) or Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Invitrogen, catalog number A11037, 1:500 dilution) for 1 h. Nuclei was stained with 0.5 μg/ml DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher Scientific) for 20 min. Slides were covered with the mounting medium (Thermo Fisher Scientific). Images were recorded with Leica confocal microscope using a 63x oil objective.
9
Immunofluorescence Analysis of Lytic Reactivation
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SLK.iBAC-GFP cells were induced with Dox (1 μg/ml) for 24 h to trigger lytic reactivation. The cells were then washed three times with PBS and fixed with 4% (w/v) paraformaldehyde (PFA) (#DF0131, LEAGENE, Beijing, China) for 10 min. Next, the fixed cells were washed with PBS for three times, permeabilized with 1% Triton X-100 for 5 min, and blocked with 10% goat serum (Antgene, Wuhan, China) for 1 hour at room temperature. After washing with wash buffer (1X PBS with 0.1% Tween 20) for three times, the cells were incubated with rabbit anti-K-RTA or mouse anti-FLAG antibodies diluted in PBS containing 1% BSA at 4°C overnight. Cells were washed with wash buffer (PBS containing 0.05% Tween-20) and then incubated with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (1:1000; Invitrogen) and Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (1:1000; Invitrogen) at room temperature for 1 hour. After washing with wash buffer and ultrapure water, the slides were mounted with DAPI Fluoromount-G mounting medium (SouthernBiotech). Finally, the images were acquired with a laser scanning confocal microscopy (Leica Stellaris 5) and processed using Image J and Leica image browser [60 (link)].
10
Immunofluorescence Visualization of Cellular Proteins
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The transfected HeLa or HEK293T cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100/PBS for 10 min, and then blocked with 5% FBS/PBS for 1 h. MxB-FLAG was detected by incubation with mouse anti-FLAG antibody (Sigma, catalog F1804-200UG, 1:1000) for 2 h, followed by incubation with Alexa Fluor 488 conjugated donkey anti-mouse secondary antibody (Invitrogen, catalog A21202, 1:500) for 1 h. TNPO1 was detected using mouse anti-TNPO1 antibody (Abcam, catalog ab10303, 1:1000) for 2 h, then with Alexa Fluor 647-goat anti-mouse secondary antibody (Invitrogen, catalog A21237, 1:500) for 1 h. KPNB1 was detected with mouse anti-KPNB1 antibody (Invitrogen, catalog MA3-070, 1:500) for 2 h and Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Invitrogen, catalog A21237,1:500) for 1 h. DNA was stained with DAPI (4=,6-diamidino-2-phenylindole). Images were recorded with a Quorum WaveFX Spinning Disk Confocal using a 63x oil objective.
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