BMDMs were washed twice, fixed by 4% cold paraformaldehyde for 30 min, permeabilized by 0.5% Triton X‐100 for 10 min, blocked by goat serum for 1 h, and incubated with anti‐ATPB (1:100, Abcam) overnight at 4°C. On the second day, BMDMs were stained with Goat anti‐mouse IgG H&L (Alexa Flour® 488, Abcam) for 1 h, followed by a second block and primary incubation (anti‐dynamic‐related protein 1 (anti‐Drp1), 1:250, Abcam; anti‐SQSTM1/p62, 1 μg/mL, Abcam). On the third day, a secondary antibody (Alexa Flour® 555, Abcam) was added to incubate with cells for 1 h. DAPI was used to stain the nucleus. Slides and stained cells were observed and photographed by a confocal microscope.
Alexa flour 555
Manufactured by Abcam
Sourced in United States
Alexa Fluor® 555 is a fluorescent dye used in various biological applications. It has an excitation maximum at 555 nm and an emission maximum at 565 nm, making it suitable for detection and visualization techniques.
Automatically generated - may contain errors
Lab products found in correlation
Alexa flour 488, by Abcam (4 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Anti sqstm1 p62, by Abcam (1 mentions)
Anti atpb, by Abcam (1 mentions)
Anti drp1, by Abcam (1 mentions)
Serpine1, by Abcam (1 mentions)
Axio imager m1 microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole dapi solution, by Thermo Fisher Scientific (1 mentions)
Col18a1, by Thermo Fisher Scientific (1 mentions)
Pecam 1, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β 3 tubulin, by Abcam (1 mentions)
4 protocols using alexa flour 555
1
Immunofluorescence Analysis of ATPB, Drp1, and p62
2
Immunofluorescence Analysis of Pancreatic Tissue
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Cells were fixed with 4% FA solutions and paraffin-embedded pancreatic tissues were deparaffinized. Fixed cells and tissues were permeabilized with Triton X-100, sequentially incubated with blocking solution and primary antibodies, including Col18a1 (Invitrogen™), Serpin E1, CD68 (Abcam Plc.), NG2 (Sigma-Aldrich), PECAM-1 (Santa Cruz Biotechnology, Inc.), and MPO (Agilent Technologies, lnc., Santa Clara, CA, USA), at 4 °C overnight, and further incubated with secondary antibodies conjugated with Alexa Flour® 488 or Alexa Flour® 555 (Abcam Plc.). DAPI (4’,6-diamidino-2-phenylindole) solution (Invitrogen™) was used to stain the nuclei. Fluorescence signals were observed and imaged using the ZEISS Axio Imager M1 microscope (Carl Zeiss, Oberkochen, Germany).
3
Immunocytochemical Profiling of Neuronal and Glial Cells
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Cultures were fixed with 4% paraformaldehyde (Alfa Aesar, MA, USA) for 10 min and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After blocking nonspecific binding sites with 3% BSA (Sigma) and 10% goat serum at room temperature for 1 h, cells were incubated overnight at 4 °C with primary antibodies against cytoskeletal marker polyclonal anti-mouse βIII-tubulin (cat# ab7751 at a 1:1000 dilution, Abcam, Cambridge, UK), mature neuronal markers; polyclonal anti-rabbit NeuN (cat# ab128886 at a 1:1000 dilution, Abcam), monoclonal anti-mouse MAP-2 (cat# ab11267 at a 1:500 dilution, Abcam), and glial cell marker polyclonal anti-rabbit GFAP (cat# ab7260 at a 1:1000 dilution, Abcam). Immune reactivity was visualized with secondary conjugate Alexa Flour 488 (cat# ab150113 and ab6717 at a 1:200 dilution, Abcam) and Alexa Flour 555 (cat# ab150078 and ab6786 at a 1:200 dilution, Abcam), which were incubated with the cells for 1 h at room temperature. DAPI was used for nuclear counterstaining. Images were captured using laser confocal microscopy (ZEISS LSM 700).
4
Immunocytochemical Characterization of PC12 Cells
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PC12 cell cultures were fixed with 4% paraformaldehyde (Alfa Aesar, Tewksbury, MA, USA) for 10 minutes and were permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature. After blocking unspecific binding sites with 3% bovine serum albumin (BSA) (Sigma, Dorset, UK) and 10% goat serum at room temperature for 1 hour, cells were incubated overnight at 4oC with primary antibodies against; polyclonal anti-rabbit Ki-67 (proliferation marker; Cat# ab 66155 at a dilution 1:1000; Abcam, Cambridge, UK), polyclonal anti-mouse βIII-tubulin (cytoskeletal marker; Cat# ab 7751 at a 1:1000 dilution; Abcam), polyclonal anti-rabbit NeuN (mature neuronal marker; Cat# ab 128886 at a 1:1000 dilution, Abcam), monoclonal anti-mouse microtubule associated protein 2 (MAP-2) (mature neuronal marker; Cat# ab 11267 at a 1:500 dilution, Abcam). Immune reactivity was visualized with secondary conjugate Alexa flour 488 (Cat# ab 150113 anti-mouse & Cat# ab 6717 anti-rabbit at a 1:200 dilution, Abcam) and Alexa flour 555 (Cat# ab 150078 anti-rabbit & Cat# ab 6786 anti-mouse at a 1:200 dilution; Abcam) for 1 hour at room temperature. DAPI was used as a nuclear counterstaining. Images were captured using laser confocal microscopy (ZEISS LSM 700, Biomedical Core Facility (BCF), Haifa, Israel).
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