Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) and 0.1% Triton X-100 for 1 h and incubated with anti-IRAK-1 (Santa Cruz Biotechnology, Inc.) at 4°C overnight. Cells were washed with PBS and incubated with goat anti-rabbit IgG-CFL 555 (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Cells were washed with PBS and stained with DAPI for 15 min at room temperature. Then, cells were washed with PBS and observed under a light microscope (magnification, ×100, BH3-MJL; Olympus Corporation).
Goat anti rabbit igg cfl 555
Manufactured by Santa Cruz Biotechnology
Goat anti-rabbit IgG-CFL 555 is a secondary antibody conjugated with CFL 555 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Bh3 mjl, by Olympus (1 mentions)
Anti irak1, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Traf6, by Cell Signaling Technology (1 mentions)
Pellino1, by Cell Signaling Technology (1 mentions)
Sc 362267, by Santa Cruz Biotechnology (1 mentions)
Axioplan 2 fluorescent microscope, by Zeiss (1 mentions)
Gal 3, by Cell Signaling Technology (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
8 well glass slides, by Merck Group (1 mentions)
Dmire2 epifluorescence microscope, by Leica (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
4 protocols using goat anti rabbit igg cfl 555
1
Immunofluorescence Staining of IRAK-1
2
Immunofluorescence Analysis of Pellino1 and TRAF6 in THP-1 Cells
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After induction model, THP-1 cell was fixed with 4 % paraformaldehyde for 15 min and then incubated with using 0.25 % Triton X100 for 15 min at room temperature. THP-1 cell was incubated with Pellino1 (1:100, 31,474, Cell Signaling Technology) and TRAF6 (1:100, 8028, Cell Signaling Technology) at 4˚C overnight after blocking with 5 % BSA for 1 h. After washing with PBS for 15 min, THP-1 cell was incubated with goat anti-rabbit IgG-cFL 555 (1:100, sc-362,272, Santa Cruz Biotechnology) or anti-mouse IgG-cFL 488 antibody (1:100, sc-362,267, Santa Cruz Biotechnology) for 2 h at room temperature and stained with DAPI for 15 min and washed with PBS for 15 min. The images of THP-1 cell obtained using a Zeiss Axioplan 2 fluorescent microscope (carl Zeiss AG, Oberkochen, Germany).
3
Gal-3 Immunofluorescence Imaging Protocol
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Cells were fixed with 4% polyformaldehyde for 20 min, blocked with 5% BSA in PBS and 0.2% Triton X-100 for 30 min. Cells were incubated with Gal-3 (1:100, Cell Signaling Technology) at 4°C overnight. Cells were incubated with goat anti-rabbit IgG-CFL 555 (1:100, Santa Cruz Biotechnology) at 37°C for 2 h after washing with PBST for 15 min and subsequently stained with DAPI in dark room for 15 min and washed with PBST for 15 min. Cells were analyzed using the Olympus BX51 fluorescence microscope.
4
Immunofluorescence Staining of Interphase and Mitotic Cells
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For imaging of interphase and mitotic cells, U2OS cells were seeded on 8-well glass slides (Millipore) and either synchronized at the G2/M transition and released as described or grown asynchronously. Immunofluorescence staining was performed by first washing cells once with 1 × PBS and fixing with a 3.7% Paraformaldehyde (PFA) in PBS solution for 10 min at room temperature. Fixed cells were permeabilized for 10 min on ice using 0.5% Triton X-100 in 1 × PBS and then blocked for 30 min in PBS-T (0.1% Tween 20 in PBS) containing 1% BSA at room temperature. Slides were then incubated with primary antibody diluted in PBS-T for 1 h at room temperature followed by three 5 min washes in PBS. After washing, the slides were then incubated with secondary antibodies, AlexaFluor 488 goat anti-mouse (Life Technologies) and goat anti-rabbit IgG-CFL 555 (Santa Cruz), in PBS-T for 1 h and washed three times with 1 × PBS. Coverslips were mounted using Fluoroshield with DAPI (Sigma) and sealed with clear nail polish. Slides were then imaged on an SP8 confocal microscope (Leica, Wetzlar, Germany) or a DM IRE2 epi-fluorescence microscope (Leica) and images processed using the Fiji distribution of ImageJ (34 (link)).
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