For immunofluorescence analysis, frozen tissue sections of breast tumor were fixed with 4% paraformaldehyde for 20 mins, washed three times with PBS, permeabilized with 0.25% Triton X-100 for 20 mins and then blocked in 5% BSA at room temperature for 1 h. For F4/80 and CXCL1 detection, tissue sections were incubated with F4/80 antibody (17-4801-80, eBioscience) and CXCL1 antibody (AF5403, Affinity) overnight at 4°C, followed by an incubation with the Alexa Fluor® 555 conjugated-anti-rat IgG (4417S, CST) and the Alexa Fluor® 488 conjugated-anti-rabbit IgG (4412S, CST) for 2 h. For ALDH1A1 detection, tissue sections were incubated with ALDH1A1 antibody (BF0220, Affinity) overnight at 4°C, followed by an incubation with the secondary antibody of Alexa Fluor® 555 conjugated-anti-mouse IgG (A21422, ThermoFisher) for 2 h. 4’, 6-diamidino-2-phenylindole (DAPI, Sigma) was used to visualize the nuclei. Fluorescence images were obtained using the LSM710 confocal microscope (Zeiss, Jena, Germany).
Alexa fluor 555 conjugated anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Alexa Fluor 555-conjugated anti-mouse IgG is a secondary antibody used to detect the presence of mouse immunoglobulin G (IgG) in various biological applications. The antibody is labeled with the Alexa Fluor 555 fluorescent dye, which emits light in the orange-red spectrum when excited. This product can be used in techniques such as immunofluorescence, flow cytometry, and Western blotting to visualize and quantify mouse IgG.
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Lab products found in correlation
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (12 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 555 conjugated anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 647 conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Mouse anti map2, by Merck Group (2 mentions)
Alexa fluor 488 conjugated anti rat igg, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Af5403, by Affinity Biosciences (1 mentions)
Bf0220, by Affinity Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsm 700 confocal fluorescence microscope, by Zeiss (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Apc conjugated anti mouse cd8a, by BioLegend (1 mentions)
Anti mneongreen, by Cell Signaling Technology (1 mentions)
Fv3000, by Olympus (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
28 protocols using alexa fluor 555 conjugated anti mouse igg
1
Immunofluorescence Analysis of Breast Tumor
2
Immunofluorescence Analysis of HEK293 Cells
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HEK293 cells were transfected with 300 ng of colFD-NL, 300 ng of Hsp47-HT, or 400 ng of empty vector; incubated with HT ligand for the same time as in the BRET assays; and fixed with 4% (w/v) paraformaldehyde for 15 min. After washing three times with PBS, the fixed cells were permeabilized using 0.1% Triton X-100 in PBS for 5 min and blocked with PBS containing 2% goat serum and 10% glycerol for 50 min at room temperature. Rabbit polyclonal antibodies recognizing Halotag (catalog no. G9281; Promega; 1/500 dilution), monoclonal anti-FLAG M2 antibody (catalog no. F1804; Sigma–Aldrich; 1/400 dilution), and monoclonal anti-PDI antibody (catalog no. ADI-SPA-891; Enzo Life Sciences, NY; 1/400 dilution) were reacted as primary antibodies for 1 h at room temperature. After washing three times with PBS, Alexa Fluor 488–conjugated anti-rabbit IgG (catalog no. A11034, Thermo Fisher; 1/400 dilution) and Alexa Fluor 555-conjugated anti-mouse IgG (catalog no. A21424; Thermo Fisher; 1/500 dilution) were reacted as secondary antibodies for 1 h at room temperature. After mounting with ProLong Gold antifade mountant (catalog no. P10144; Thermo Fisher), fluorescent signals were analyzed using an LSM 700 confocal fluorescence microscope (Carl Zeiss) with an appropriate setup of lasers, beam splitters, and filters for Alexa Fluor 488, Alexa Fluor 555, and HT ligand 618.
3
Multimodal Antibody Characterization
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The following antibodies were used for Western blot, confocal microscopy and flow cytometric analyses: Anti-Flag®BioM2 (mouse, #F9291, Merck), Anti-mNeonGreen (rabbit, #53061, Cell Signalling Technologies, Danvers, MA, USA), biotin-conjugated anti-GAPDH (goat, #A00915, Genscript, Piscataway, NJ, USA), anti-human cleaved caspase-8 (mouse, #sc-5263, Santa Cruz Biotechnologies), anti-human granzyme B (mouse, #sc-8022, Santa Cruz Biotechnologies), anti-human HSP 70 (mouse, #sc-24, Santa Cruz Biotechnologies), AlexaFluor®647-conjugated anti-rabbit IgG (goat, #A32733, Thermo Fisher Scientific), AlexaFluor®555-conjugated anti-mouse IgG (goat, #A21422, Thermo Fisher Scientific), APC-conjugated anti-mouse CD8a (rat, #100712, BioLegend, San Diego, CA, USA) and DyLight®549-conjugated streptavidin (#A21837, Thermo Fisher Scientific).
4
Immunostaining of Primary Hippocampal Neurons
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Primary hippocampal neurons were fixed with chilled methanol for 8 min at − 20 °C. Hippocampal tissue sections and primary neurons were permeabilized with 0.3% Triton X-100 for 5 min. The tissues and cells were blocked with 10% bovine serum albumin (BSA) and incubated overnight at 4 °C with the following primary antibodies: mouse anti-MAP2 (Sigma-Aldrich, M9942), rabbit anti-PSD95 (Abcam, ab18258), rabbit anti-Synaptophysin (Abcam, ab32127), mouse anti-GFAP (Millipore, MAB360), rat anti-CD11b (Abcam, ab8878), mouse anti-Nlgn3 (Santa Cruz, SC-271880). After treatment with primary antibodies, the tissues and cells were incubated with secondary antibodies conjugated with Alexa Fluor 488-conjugated anti-rabbit IgG (ThermoFisher, A11008), Alexa Fluor 555-conjugated anti-mouse IgG (ThermoFisher, A21422), or Alexa Fluor 488-conjugated anti-rat IgG (ThermoFisher, A11006) for 1.5 h at room temperature. Immunostained tissues and cells were mounted with VECTASHIELD Antifade mounting medium with DAPI (H-1200; Vector Laboratories). Alexa Fluor 488 (excitation, 488 nm; emission, 520 nm) and Alexa Fluor 555 (excitation, 561 nm; emission, 568 nm)-labeled tissues and cells were imaged using a confocal microscope (FV3000, Olympus).
5
Fluorescent Protein Labeling and Membrane Disruption
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Alexa Fluor 555-conjugated anti-mouse IgG, Alexa Fluor 488-conjugated anti-mouse IgG, and Alexa Fluor 488-conjugated anti-rabbit IgG were from Thermo Fisher Scientific. Phospholipase C (PLC) inhibitor U73122 and methyl-β-cyclodextrin (MβCD) were from Sigma/Merck.
6
Visualizing Transcription Factor Localization
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SW1353 cells were transfected with Flag-Zfhx4, Venus-Osterix, or Venus-Runx2 using the X-tremeGENE 9. After 48 h, cells were washed twice with PBS and fixed with 4% buffered paraformaldehyde (WAKO, Osaka, Japan) for 20 min. After treatment with 0.2% Triton X-100 in PBS for 5 min, cells were blocked with 1% BSA in PBS for 1 h, incubated with an anti-Flag (WAKO) antibody at room temperature for 2 h, and then incubated with Alexa Fluor 555-conjugated anti-mouse IgG (1:500, A21424; Thermo Fisher Scientific) for 30 min. Nuclear staining was performed using Vectashield with 4′,6-diamidino-2-phenylindole (Vector, Burlingame, CA, USA). Samples were visualized using a Leica TCS SP8 confocal microscope (Leica Microsystems).
7
Immunofluorescence Imaging of Cellular Proteins
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For the immunofluorescence assay, cells were incubated with primary antibodies overnight at 4℃, followed by incubation with Alexa Fluor 555-conjugated anti-mouse IgG (A32794, Thermo Fisher, Massachusetts, USA) and Alexa Fluor 647-conjugated anti-rabbit antibody (ab150075, Abcam, Cambridge, UK). Confocal imaging was performed using a confocal laser scanning microscope (LSM880, Carl Zeiss, Germany).
8
Cryosectioning and Immunostaining of Tissue
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Cells attached to a glass coverslip or tissue section in O.C.T. compound (Sakura Finetek) were used for immunostaining. Tissue sections were prepared at 10 µm thickness using a cryostat (NX70, Leica) and dried at 37 °C for 10 min. The samples were washed twice in 2 mL PBS (–) and fixed in 2% paraformaldehyde (Nacalai Tesque) for 5 min. The samples were then dehydrated with acetone (Nacalai Tesque) for 5 min. Permeabilization was performed in 50 µL 0.5% Triton X-100 (Nacalai Tesque) for 5 min. Samples were then blocked with 10% goat serum (143-06561, Wako) in BlockAce solution (KAC Co., Ltd.) at room temperature for 1 h, followed by washing in 0.1% BSA/PBS (Sigma) twice for 5 min each. Primary antibody (for DNA DSBs analysis, anti-γH2AX, 1:50, Biolegend, 613402; for immunohistochemistry of thymus, anti-K8, 1:50, Biolegend, 904804, and anti-K5, 1:50, Biolegend, 905504) was incubated at 4 °C overnight under humid conditions. The secondary antibody (Alexa Fluor 555 conjugated anti-mouse IgG, Thermo Fisher, A21425; Alexa Fluor 647 conjugated anti-rabbit IgG, Thermo Fisher, A21246) was incubated at room temperature for 1 h. Nuclei were stained with Hoechst33342 (1:1000; KV072, Wako) for 3 min before imaging with a fluorescent microscope (EVOS® FL, Invitrogen). Intensity of γH2AX signals were accessed using ImageJ software46 (link).
9
Immunofluorescence Staining of SMAD3 and α-SMA
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Immunofluorescence staining was performed to detect the expression of mothers against decapentaplegic homolog 3 (SMAD3) protein and α-smooth muscle actin (α-SMA). Briefly, NRK-49F cells were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized for 5 min using 0.1% Triton X-100. After washing with PBS, the cells were blocked with 5% bovine serum albumin (Merck KGaA) in PBS containing Tween-20 at room temperature for 1 h, then incubated with primary mouse anti-α-SMA (1:400, A5228; Merck KGaA) and rabbit anti-phosphorylated-SMAD3 (1:200, ab52903; Abcam, Cambridge, MA, USA) antibodies overnight at 4°C. Subsequently, the cells were incubated with Alexa Fluor 555-conjugated anti-mouse IgG (1:500, A021422; Thermo Fisher Scientific, Inc.) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, ab150077; Abcam) antibodies at room temperature for 1 h, then stained with DAPI at room temperature for 10 min prior to fluorescence microscopy.
10
RF-EMF Exposure Effects on Hippocampal Neurons
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Hippocampal neurons were exposed to 1760 MHz RF-EMF for 5 h/day from 0 to 9 days in vitro (DIV). Hippocampal neurons were prepared as previously described [63 (link)]. Hippocampal neurons were fixed with cooled methanol for 10 min at −20 °C and permeabilized with 0.3% Triton X-100 for 5 min. The cells were blocked with 10% BSA and incubated for 16 h at 4 °C with the following primary antibodies: mouse anti-MAP2 (Sigma-Aldrich, St. Louis, MI, USA), rabbit anti-PSD95 (Abcam, Cambridge, UK), rabbit anti-AMPAR1 (GluR1) (Abcam, Cambridge, UK), rabbit anti-NMDAR1 (NR1) (Abcam, Cambridge, UK), and rabbit anti-BDNF (Abcam, Cambridge, UK). The cells were then incubated in Alexa Fluor 488-conjugated anti-rabbit IgG (ThermoFisher, Rockford, IL, USA) and Alexa Fluor 555-conjugated anti-mouse IgG (ThermoFisher, Rockford, IL, USA) for 1 h 30 min at room temperature. After washing in PBS, coverslips were mounted with VECTASHIELD Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA). Alexa Fluor 488 (excitation, 488 nm; emission, 520 nm) and Alexa Fluor 555 (excitation, 561 nm; emission, 568 nm)-labeled neurons were imaged using an FV3000 confocal microscope (FV3000, Olympus, Tokyo, Japan).
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