HCT116 cells grown on glass coverslips were transfected with control siRNA or siHsp90, respectively. At 48 hours post-transfection, the cells were fixed with 4% ice-cold paraformaldehyde at 4 °C for 20 min and then permeabilized with PBS with 0.5% Triton X-100 for 10 min at room temperature (RT), then washed, and blocked with 10% goat serum in Phosphate-Buffered Saline (PBS) for 45 minutes at RT. Cells were then incubated overnight at 4 °C with the mouse anti-Hsp90 (1:300; Abcam, MA, USA) or rabbit anti-HMGA2 (1:300; Santa Cruz Biotechnology, TX, USA). After washing, the cells were incubated at RT for 1.5 hours with Alexa-Fluor-546-conjugated goat anti-mouse IgG secondary antibody (1:500) (Invitrogen, Carlsbad, CA, USA) or Alexa-Fluor-488-conjugated goat anti-rabbit IgG secondary antibody (1:500) (Invitrogen, Carlsbad, CA, USA). After 3 washes, cells were mounted on glass slides in Mount medium containing DAPI (4, 6 diamidino-2-phenylindole;Polysciences) (Vector Laboratories, CA, USA). The images were examined on an Olympus FV1000 confocal microscope (Olympus Corp., Tokyo, Japan).
Alexa fluor 546 conjugated goat anti mouse igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa-Fluor-546-conjugated goat anti-mouse IgG secondary antibody is a fluorescently labeled antibody that binds to mouse immunoglobulin G (IgG) antibodies. It is used for detection and visualization of target proteins in various immunoassay techniques, such as immunofluorescence microscopy and Western blotting.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Mouse anti hsp90, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Cholesterol assay kit, by Abcam (1 mentions)
Lsm 780 nlo, by Zeiss (1 mentions)
Silica gel blue, by Samchun (1 mentions)
Fv200 microscope, by Olympus (1 mentions)
Fv10 asw 3.0 viewer software, by Olympus (1 mentions)
Fluoview fv1000 laser scanning confocal imaging system, by Olympus (1 mentions)
5 protocols using alexa fluor 546 conjugated goat anti mouse igg secondary antibody
1
Immunofluorescence Analysis of Hsp90 and HMGA2
2
Engineered Skeletal Muscle Tissue Immunostaining
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Engineered human skeletal muscle tissues were fixed by treatment with 4% (vol/vol) paraformaldehyde for 15 min (16% stock diluted in PBS; Electron Microscopy Sciences) and permeabilized with 0.05% (vol/vol) Triton X-100 in PBS at room temperature for 5 min. The samples were then washed with PBS before incubation with primary antibodies. The samples were incubated with a primary antibody solution consisting of a 1:200 dilution of monoclonal anti-SAA antibodies (A7811; clone EA-53; Sigma-Aldrich) in PBS for 2 h at room temperature. After rinsing three times with room temperature PBS, the engineered tissues were incubated with a secondary solution consisting of 1:200 dilutions of Alexa Fluor 546–conjugated goat anti–mouse IgG secondary antibody (Invitrogen), Alexa Fluor 488–conjugated phalloidin (A12379; Invitrogen), and DAPI (D1306; Invitrogen) for 1 h at room temperature. Fluorescence microscopy was performed using a laser-scanning confocal microscope (ZEISS) using the enhanced contrast Plan-Neofluar 40×/1.3 oil differential interference contrast M27 objective at room temperature.
3
Cholesterol Trafficking in HeLa Cells
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HRV1B-infected or uninfected HeLa cells (2 × 103) were treated with ICZ (10 μM), cholesterol/CD (20 μg/ml), and the cholesterol synthesis inhibitor U18666A (1.25 μM) (included in the cholesterol assay kit from Abcam, Cambridge, UK). Cells were fixed with a cell-based assay fixative solution for 10 min and stained with filipin III for 30–60 min at room temperature according to the manufacturer’s protocol. An antibody against human LAMP-1, a late endosome/lysosome marker, was purchased from Biolegend (San Diego, CA, USA). Alexa Fluor 546-conjugated goat anti-mouse IgG secondary antibody was purchased from Invitrogen Life Technologies. Cells were visualized by confocal microscopy (LSM780 NLO; Carl Zeiss, Jena, Germany).
4
Immunofluorescence Assay for Malaria Parasites
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Blood smears were prepared for IFA as described previously [19 (link)]. Slides smeared with schizont enriched blood were fixed with ice-cold acetone for 3 min, dried, and stored at -80˚C until use. The frozen slides were thawed on silica gel blue (Samchun Chemical, Pyeongtaek, Korea) and blocked with 5% BSA in PBS at 37˚C for 30 min. The slides were incubated with 1:100 dilutions of primary antibodies (mouse anti-Pv92 and rabbit anti-PvMSP1-19) at 37˚C for 1 hr. The slides were then stained with Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 546-conjugated goat anti-mouse IgG secondary antibodies (Invitrogen) and the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen) at 37˚C for 30 min. The slides were mounted in ProLong Gold antifade reagent (Invitrogen) and visualized under oil immersion using a confocal laser scanning FV200 microscope (Olympus, Tokyo, Japan). Images were captured using the FV10-ASW 3.0 viewer software.
5
Localization of PvMSP1-19 and PkMSP1P-19
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To confirm the localization of PvMSP1-19 and PkMSP1P-19, blood smears for indirect immunofluorescence assay (IFA) were prepared as described previously (Li et al., 2012 (link)). Slides smeared with P. knowlesi schizont-enriched blood were fixed with 4% paraformaldehyde and blocked with 5% skim milk in PBS. Rabbit anti-PkMSP1-19 and mouse anti-PkMSP1P-19 diluted in 1:50 were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 546-conjugated goat anti-mouse IgG secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA) and 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen Life Technologies) were used for secondary antibody staining. The slides were mounted with ProLong Gold Antifade reagent (Invitrogen) and visualized under immersion oil using the FluoView® FV1000 Laser Scanning Confocal Imaging System (Olympus, Tokyo, Japan) equipped with a 60× oil objective. Images were captured using the FV10-ASW 3.0 Viewer software (Olympus). The fluorescence graphic containing more than 500 pixels was calculated by ImageJ (NIH, Rockville, MD).
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