For the NF-κB translocation assay, THP-1-derived macrophages were induced with high glucose in the presence or absence of rapamycin. The cells were fixed with 95% acetone for 30 min and washed with PBS for an additional 5 min. Immunostaining was performed using rabbit anti-p65 antibody (1:350 dilution), followed by Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:3,000 dilution, Abcam, Cambridge, UK). After washing, the cells were stained with 20 μL Hoechst 33258 (Invitrogen, USA) for 3 min. Then, slides were washed and mounted with glycerol jelly mounting medium and photographed with a fluorescence microscope.
Alexa fluor 488 conjugated goat anti mouse igg antibody
Manufactured by Abcam
Sourced in United Kingdom
Alexa Fluor 488-conjugated goat anti-mouse IgG antibody is a secondary antibody that targets mouse immunoglobulin G (IgG) and is conjugated to the Alexa Fluor 488 fluorescent dye. It can be used to detect and visualize mouse IgG in various immunoassays and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Golm1, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dmi4000b, by Leica (1 mentions)
Cell culture dish, by NEST Biotechnology (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
4 protocols using alexa fluor 488 conjugated goat anti mouse igg antibody
1
NF-κB Translocation Assay in THP-1 Macrophages
2
Immunofluorescence Staining of GOLM1 and TRA1-85
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Cells were cultured on coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.4%Triton X-100, blocked with 5% bovine serum albumin, and incubated with primary antibody against GOLM1 (1:200; Abcam) or TRA1–85 (1:200; R&D Systems) at 4 °C overnight. Primary antibody was subsequently detected with an Alexa Fluor 594 conjugated goat anti-rabbit IgG antibody (1:800; Abcam) or Alexa Fluor 488 conjugated goat anti-mouse IgG antibody (1:800; Abcam) respectively. The cytoskeleton was visualized through staining with anti-stain 488 phalloidin (Cytoskeleton; Denver, CO, USA) according to manufacturer’s instructions. Cell nuclei were stained with DAPI (Sigma-Aldrich; Hamburg, Germany). Images for analysis were obtained under fluorescence microscopy (Leica; Wetzlar, Germany).
3
ATRA Modulates NF-κB Activation in IPEC-J2 Cells
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IPEC-J2 cells were cultured in a 12-well cell culture plate and infected with TGEV at MOI of 1 for 1 h. Afterwards, the cells were washed with PBS and cultured with 80 μM ATRA for 36 h. After cell treatment, IPEC-J2 cells were fixed with cold 4% paraformaldehyde for 15 min and permeated with 0.05% Triton X-100 for 15 min at room temperature. Then, the IPEC-J2 cells were incubated with primary anti-NF-κB p65 mouse antibodies overnight at 4°C (1:100 dilution, Cell signaling Technology, USA). The cells were then washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Abcam, Shanghai, China) for 2 h at room temperature in the dark. After washing with PBS, the cells were mounted using Vectashield Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Shanghai, China). Images were captured using a fluorescence microscope (Leica DMI 4000 B).
4
Visualization of Recombinant Protein Binding
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Binding of recombinant proteins to PBLs was detected by immunofluorescence microscopy as reported previously (44 (link)) with slight adjustments. Briefly, Japanese flounder PBLs were resuspended in L-15 medium to 1×107 cells/ml, and the cells were added to cell culture dish (NEST, USA) for 4 h to allow the cells to settle. The cells were then blocked with 5% skim milk and incubated at 22°C for 1 h. After washing with PBS, 1 μM rPoCXCL10, 1 μM rPoCXCL10M, or PBS were added to the dish and incubated at 22°C for 2 h, followed by washing with PBS for three times. The cells were treated with mouse anti-Flag antibody (ABclonal, Wuhan, China) at 1/1,000 dilution for 1h and then treated with Alexa Fluor® 488-conjugated goat anti-mouse IgG antibody (Abcam, UK) at 1/2,000 dilution for 1 h, followed by washing with PBS for three times. The cells were fixed with 4% paraformaldehyde (Biosharp, Shanghai, China) for 30 min and washed as above, then the cells were stained with 1, 1′-dioctadecyl-3, 3, 3, 3′-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen, Carlsbad, CA, USA) and 4’, 6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). The cells were then observed with a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
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