Human PDAC tissues were fixed in 10% buffered formalin solution. Paraffin-embedded sections were deparaffinized, and antigen retrieval was performed by microwave treatment. The sections were incubated with the first antibodies at 37°C for 1 h followed by the application of a second antibody, either Alexa594-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) or Alexa488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific). The study using the tissue specimens was approved by the Research Ethics Committee in Kawasaki Medical School and Hospital (approved No. 3105). Informed consent was obtained from each patient for the use of these materials.
Alexa 488 conjugated goat anti mouse igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 488-conjugated goat anti-mouse IgG antibody is a secondary antibody used to detect and visualize mouse immunoglobulin G (IgG) in various immunoassays and cellular imaging applications. The Alexa Fluor 488 fluorescent dye is conjugated to the antibody, allowing for green fluorescent detection.
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Lab products found in correlation
Alexa 594 conjugated goat anti rabbit igg antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lab tek chamber slide system, by Thermo Fisher Scientific (1 mentions)
Anti c myc antibody clone 9e10, by Merck Group (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Dmi3000b inverted microscope, by Leica (1 mentions)
Facscalibur, by BD (1 mentions)
Protein block, by BioGenex (1 mentions)
Citric acid based antigen unmasking solution, by Vector Laboratories (1 mentions)
Fluoroshield containing dapi, by Merck Group (1 mentions)
Guinea pig anti insulin, by Thermo Fisher Scientific (1 mentions)
Observer z1 microscope, by Zeiss (1 mentions)
Lsm510 fluorescence microscope, by Zeiss (1 mentions)
Mouse monoclonal anti α sma antibody, by Merck Group (1 mentions)
A2547, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Dutscher (1 mentions)
Rabbit anti β actin polyclonal antibody, by ABclonal (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cryostar nx70 cryostat, by Thermo Fisher Scientific (1 mentions)
13 protocols using alexa 488 conjugated goat anti mouse igg antibody
1
Immunofluorescence analysis of PDAC tissues
2
Immunocytochemistry of Various Cell Types
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For immunocytochemistry (ICC), 50 000 target cells (HBVP, PBVP, hMscTert, ASF2, HBMEC and HMEC-1) were seeded in each well of an 8-well Nunc Lab-Tek Chamber Slide System and incubated overnight for attachment. The cells were rinsed with dPBS, fixed in 2% PFA for 15 min at RT and subsequently blocked in 2% (w/v) MPBS for 1.5 h at RT. For staining on HBVP only, the primary antibodies were diluted to a final concentration of 40 μg/ml in 2% MPBS and incubated overnight at 4°C. The next day, the wells were washed 2 × 5 min in PBS and incubated with a secondary anti c-Myc antibody (clone 9E10, Sigma-Aldrich) diluted 1:500 in 2% (w/v) MPBS, for 2 h at RT. The cells were washed 3 × 5 min in PBS and the secondary antibody was detected by an Alexa 488 conjugated Goat anti Mouse IgG antibody (Thermo Fisher Scientific). For assessment of antibody specificity though ICC on different cell types, the primary antibodies were diluted to a final concentration of 60 μg/ml in 2% MPBS and incubated overnight at 4°C. The wells were washed 2 × 5 min in PBS and the primary antibodies were detected by a monoclonal mouse Anti-c-Myc-Cy3 antibody (clone 9E10, Sigma-Aldrich) diluted 1:100. For both experiments, cell nuclei were stained using VECTASHIELD Mounting Medium with DAPI (Vector Labs, USA). Fluorescent images were obtained with a Leica DMI3000 B inverted microscope (Leica Microsystems, Germany).
3
BrdU Incorporation Assay in PBMC
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Fixed PBMC (1 × 106 cells) were depurinated for 30 minutes in 2N HCl, 0.5% Triton X‐100 in PBS, blocked with 1% FBS, and reacted with an anti–BrdU antibody (B44; BD Bioscience) or control mouse IgG antibodies. Samples were incubated with an Alexa 488‐conjugated goat anti–mouse IgG antibody (Thermo Fisher Scientific) with propidium iodide (5 µg/mL) and analyzed using a FACS Calibur (BD Bioscience).8 The gated area of FTD‐negative PBMC and the boundary between FTD‐positive and FTD‐negative PBMC were determined in each sample using a control mouse antibody against IgG.
4
Immunofluorescence Staining of CD105 and Insulin
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Cells were cultured to 70–80% confluence and dissociated into a single-cell suspension using TrypLE as described [33 (link)]. Cells and pancreatic tissue were then fixed in 10% cold formalin, prepared in a paraffin block, and sectioned. Antigen retrieval was performed using a citric acid-based antigen unmasking solution (Vector, pH 6.0). Sections were treated with protein block (Biogenex, Fremont, CA) to reduce background signal, followed by incubation with mouse anti-CD105 antibody (ready to use; Biogenex) and ALEXA 488-conjugated goat anti-mouse IgG antibody (1:200 dilution; ThermoFisher). Guinea pig anti-insulin (ThermoFisher) and ALEXA 647-conjugated goat anti-Guinea pig IgG antibodies (1:200 dilution; ThermoFisher) were used for pancreatic tissue staining only. Fluoroshield™ containing DAPI (Sigma Aldrich St. Louis, MO) was used to stain nuclei. Image acquisition was done using an Observer Z1 microscope (Carl Zeiss), with the objective lens set at 20×. Image processing was done using the Zen 2.0 software.
5
Immunofluorescence Staining of α-SMA
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HIFs (1 × 104 cells/well) were seeded onto chamber slides (#30,108, SPL Life Sciences, South Korea) and exposed to TGF-β1 (5 ng/ml) co-incubated with or without TRG/RSG for 24 h, after which the cells were fixed with 4% paraformaldehyde (PFA). After permeabilization with 0.1% Triton-×100 in 1 × PBS and blocking with 5% bovine serum albumin (BSA), the cells were incubated with mouse monoclonal anti-α-SMA antibody (1:500, A2547, Sigma-Aldrich) at 4 °C overnight followed by treatment with goat Alexa488-conjugated anti-mouse IgG antibody (A11029, Thermo Fisher Scientific, Rockford, IL) at room temperature for 2 h. For the staining of actin filaments (F-actin), the cells were incubated with rhodamine-phalloidin (1:200, R415, Invitrogen). After washing, the nuclei were counterstained by Hoechst 33,342 (B2261, Sigma-Aldrich) according to manufacturer’s instructions. The stained sections were immediately covered and examined under a Zeiss LSM510 fluorescence microscope or Zeiss LSM880 confocal laser scanning microscope.
6
Culturing and Immunostaining of Human Hepatoma Cells
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Human hepatoma Huh7 cells (a generous gift from Dr V. Lotteau, CIRI, Lyon) were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (Dutscher, Brumath, France) and antibiotics (PAN Biotech Dutscher, Brumath, France) at 37 °C under a 5% CO2 atmosphere. The mouse anti-DDDDK tag (FLAG) antibody (Abcam, Cambridge, UK) in PBS, rabbit DENV-2 NS1 polyclonal antibody PA5-33207 (anti-NS1 pAb), and goat Alexa 488-conjugated anti-mouse IgG antibody were purchased from Thermo Fisher Scientific (Les Ulis, France) in PBS. The mouse anti-flavivirus NS1 antibody [D/2/D6/B7] (anti-NS1 mAb) and anti-mouse IgG HRP-conjugated secondary antibodies were purchased from Abcam (Cambridge, UK). The rabbit anti-β actin polyclonal antibody was purchased from ABclonal (MA, Woburn, USA).
7
Cell Surface Protein Expression Analysis
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Cells were plated in triplicate at 6 × 105 per well in 6 well plates. They were grown for 24 h until confluent. Cells were detached with Trypsin, quenched with 10% FBS-DMEM and pelleted by centrifugation in 1.5 ml microfuge tubes at 350 g, 4°C for 3 minutes. Cells were resuspended at 5 × 105 per 50 μl in 10% NGS (normal goat serum) FACS buffer (1% (w/v) BSA, 1 mM EDTA, 25 mM HEPES-KOH pH 7.4 in PBS). Cells (5 × 105) were incubated in 10% NGS FACS buffer for 15 min on ice, centrifuged and the pellets resuspended in 10% NGS FACS buffer containing either 10 μg/ml monoclonal mouse anti-CDCP1 clone CUB1 or 40 μg/ml monoclonal mouse anti-CD9 clone ALB6 or corresponding isotype-matched control antibodies. Cells were incubated for 30 min on ice, centrifuged and washed in 50 μl FACS buffer. Cells were resuspended in 50 μl 10% NGS FACS buffer containing 2 μg/ml Alexa 488-conjugated goat anti-mouse IgG antibody (Invitrogen A-11029) and incubated for 30 min on ice. Cells were centrifuged and pellets resuspended in 500 μl PBS. Cells were analysed on a Becton Dickinson FACScalibur flow cytometer using CELLQuest software.
8
Histological Analysis of TA and DIA Muscles
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The TA and DIA muscles were sectioned at 8 μm using a CryoStar NX70 cryostat (Thermo Fisher) and stained with H&E or Sirius red. IgG staining was performed to detect degenerating fibers as previously described (54 (link)). In brief, the cryosections were preincubated with 5% BSA in PBS for 1 h, followed by incubation with Alexa 488-conjugated goat anti-mouse IgG antibody (A-11029, Invitrogen, Carlsbad, CA). Fluorescence images were obtained using a BZ-X810 fluorescence microscope (Keyence).
9
Immunostaining of DENV2-infected Huh7.5.1 and BHK-21 cells
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Huh7.5.1 cells were plated onto glass coverslips, infected with DENV2 strain 16681 for 48 h, washed in PBS buffer, pH 7.4, and fixed for 30 min with a freshly prepared 4% formaldehyde solution. The cells were permeabilized for 15 min with 0.6% saponin in PBS buffer, pH 7.4. The coverslips were incubated in blocking solution containing 1.5% BSA in PBS and then incubated for 1 h with purified anti-NS3 helicase polyclonal antibodies (made in-house) and anti-GAPDH polyclonal antibodies (Abcam, USA) diluted 1:100 in blocking solution. The cells were washed and incubated for 45 min with an Alexa 488-conjugated goat-anti-mouse IgG antibody or with an Alexa 546-conjugated goat anti-rabbit IgG antibody (Invitrogen, USA) diluted 1:400 in blocking solution. The cells were subsequently incubated with 5 µM 4′,6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 10 min at room temperature. The slides were mounted in N-propyl gallate and observed using a Leica TCS SP5 confocal microscope. All images were collected with LAS AF Lite 2.6 software (Leica Microsystems, USA). The same protocol was used for the pcDNA3.1- and pcDNA3.1-NS3-transfected BHK-21 cells.
10
Nuclear Translocation of p65 in Macrophages
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Immunocytochemistry was performed to assess the effects of auranofin on the nuclear translocation of p65 in the Raw264.7 macrophage cell line. The Raw264.7 cells treated with vehicle or 3 μM auranofin were fixed with 4% paraformaldehyde for 15 min, washed three times with 0.2% Triton X-100 in PBS for 10 min, blocked with 1% horse serum in PBS, and incubated with monoclonal anti-p65 antibody followed by Alexa-488 conjugated goat anti-mouse IgG antibody (A-21202, Invitrogen, Carlsbad, CA). Cells were counterstained with prolong gold antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Scientific, Rochester, USA).
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