TK6 cells (0.5 million/ml) were exposed to IPMS (750μM) for 24 hours. Cells were then fixed using 2% formaldehyde / PBS for 20 min, washed in PBS, and spotted on to double frosted microscopic glass slides (Fisher Scientific) at a concentration of 2 million/ml. Blocking was achieved using 5% (w/v) bovine serum albumin (BSA) in PBS-TT for 30 min. Cells were then incubated with 1:500 rabbit polyclonal anti-53BP1 antibody (Bethyl Laboratories, Montgomery, TX) and mouse γH2AX antibody (Millipore Corporation, CA, USA) in 1% BSA/PBS-TT for overnight at 4oC. Cells were then washed in PBS, incubated in 1:250 anti-mouse Alexa Fluor 555 (Life Technologies) and Alexa Fluor 488 antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1h. After washing with PBS, slides were mounted with a cover slip using Vectashield with DAPI (Vector Laboratories) and sealed using nail polish. Slides were analyzed using confocal microscopy (Zeiss CLSM 700). Optical sections through the nuclei were captured at 0.5 μm intervals, and the images were obtained by maximum projection of the individual sections.
Alexa fluor 488 antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 488 antibody is a fluorescent dye conjugated to an antibody molecule. It is designed for use in various fluorescence-based applications, such as flow cytometry, fluorescence microscopy, and immunoassays. The Alexa Fluor 488 dye exhibits bright green fluorescence when excited by a 488 nm light source.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Clsm 700, by Zeiss (1 mentions)
Mouse γh2ax antibody, by Merck Group (1 mentions)
Anti 53bp1 antibody, by Fortis Life Sciences (1 mentions)
Streptavidin alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Thunder imaging system, by Leica (1 mentions)
780 confocal laser scanning microscope, by Zeiss (1 mentions)
Apotome 2 microscopy, by Zeiss (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Zeba spin column, by Thermo Fisher Scientific (1 mentions)
Nab protein a g spin kit, by Thermo Fisher Scientific (1 mentions)
Spectrofluorometer, by Horiba (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Anti neun antibody, by Cell Signaling Technology (1 mentions)
Nis elements software, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
6 protocols using alexa fluor 488 antibody
1
Quantification of DNA Damage Markers
2
Perineuronal Net Staining in Brain Sections
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(See Supplementary). In brief, 30 μm brain sections were stained with WFA, a lectin that binds N-acetylgalactosamines-β1 residues of PNN. Fluorescent staining was performed using biotin-conjugate WFA (1:1000; #L1516, Sigma-Aldrich, St. Louis, MO) and secondary antibodies (Streptavidin Alexa Fluor 488, 1:200, #S32354, Invitrogen, Carsband, CA). We performed double staining for WFA and PV (monoclonal rabbit anti-PV antibodies; 1:1000, Swant, Switzerland; #PV27) using secondary Alexa Fluor 488 antibodies (1:200) and donkey anti rabbit Cy3 secondary antibodies (1:200, #711-165-152, Jackson ImmunoResearch, Cambridge, UK). Sections were examined with a ZEISS 780 confocal laser scanning microscope, a Zeiss Carl Apotome2 microscopy (Zeiss, Gottingen, Germany) and a Thunder Imaging System by Leica Microsystem (Wetzlar, Germany) and processed with ZEN software and/or LAS X software. Cell counting was performed unilaterally in three coronal sections (+0.98, +0.02, and −1.06 mm from Bregma), at 10x magnification from an observer who was aware of the treatment.
3
CHOV-GP Specific IgG Antibody Assay
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Serum specific IgG antibodies against CHOV-GP were detected by a cell-based assay and flow cytometry. Briefly, CHOV GPs-293T were used to detect binding of purified IgGs (10 µg/ml for IgG determination) from subjects (15 acute and 15 convalescent, 33 seropositive and 14 healthy donors). Polyclonal IgG was isolated from serum using NAb™ Protein A/G Spin Kit (Thermo Scientific) followed by desalting using Zeba spin columns (Thermo Scientific) according to manufacturer´s instructions.
Reactive cells were detected with Alexa Fluor 488 anti-human IgG antibody (Jackson Immuno Research). For IgG subclass determination, CHOV GPs-293T cells were incubated with sera (1/1000), washed, stained with anti-human -IgG1, -IgG2, -IgG3, or -IgG4 mouse antibody (Southern Biotech) and labeled with anti-mouse Alexa Fluor 488 antibody (Jackson ImmunoResearch). All antibody stainings were incubated for 1 hour at 4°C. The levels of CHOV GP-specific antibody binding were analyzed by flow cytometry (BD LSRFortessa X-20).
Reactive cells were detected with Alexa Fluor 488 anti-human IgG antibody (Jackson Immuno Research). For IgG subclass determination, CHOV GPs-293T cells were incubated with sera (1/1000), washed, stained with anti-human -IgG1, -IgG2, -IgG3, or -IgG4 mouse antibody (Southern Biotech) and labeled with anti-mouse Alexa Fluor 488 antibody (Jackson ImmunoResearch). All antibody stainings were incubated for 1 hour at 4°C. The levels of CHOV GP-specific antibody binding were analyzed by flow cytometry (BD LSRFortessa X-20).
4
Antibody Characterization via FRET Assay
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Both desired and non-desired antibodies were harvested through centrifugation of 9e10 and 7R2/A4 hybridoma cultures respectively. Supernatant rich in antibodies was filtered through 0.2 μm size membrane and mixed with 16 μg/mL polyclonal goat anti-mouse (Alexa Fluor®-488) antibody (Jackson ImmunoResearch) and 8 μg/mL Alexa Fluor®-647 labeled c-myc peptide (JPT Peptide Technologies GmbH), resulting in the positive and negative sample mix. The control sample was prepared by mixing both detection antibody and peptide with a fresh complete RPMI-1640 cell growth medium. All samples were then analyzed on a spectrofluorometer (Horiba), using an excitation wavelength of 488 nm, recording the emission spectrum of both FRET donor and acceptor from 550 to 750 nm (Figure 1 ).
5
Quantification of Motor Neurons in Spinal Cord
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For motor neuron immunostaining and quantification, spinal cord sections from mice sacrificed at PD91 were used and prepared via slightly modified methods from our previous publications [24 (link),25 (link)]. In brief, 12–15 sections per mouse across 400 µm of the lumbar spinal cord were selected and incubated with primary rabbit monoclonal anti-NeuN antibody (1:500, Cat. #12943, Cell Signaling, Inc., Beverly, MA, USA) to label neurons. After incubation with secondary goat anti-rabbit Alexa Fluor 488 antibody (1:200, Cat. #111-545-045, Jackson Immunoresearch Laboratories, West Grove, PA, USA), neurons in the ventral horn were imaged using a Nikon Eclipse Ti epifluorescent microscope and Nikon NIS-Elements software (Nikon Instruments, Inc., Melville, NY, USA). Nuclei were labeled with Hoechst 33342 (bisbenzimide H 33342 trihydrochloride, Cat. #B2261, Sigma-Aldrich, St. Louis, MO, USA). For quantification of ventral horn MN, only large morphologically-intact neurons with a diameter ≥25 µm and distinct cell nucleus were counted per section. Small or injured neurons in the ventral horn with fragmented soma were not counted.
6
Immunofluorescence Staining of Cellular Organelles
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Cells were fixed with 4% formaldehyde for 15 min at room temperature, washed and blocked for 1 h in 5% BSA (Millipore, Bedford, MA, USA) with 0.1% Triton-X 100 in PBS. Then, cells were incubated overnight with primary antibodies against LAMP1 (D2D11, 1:500, Cell Signaling Technology, Danvers, MA, USA) and TUJ1α (T8660,1:1000, Sigma-Aldrich Aldrich, Saint Louis, MO, USA) at 4 °C. Followed by a 2 h incubation at room temperature with donkey anti-rabbit Alexa Fluor 594 antibody (711-585-152, 1:500, Jackson ImmunoResearch, West Grove, PA, USA) and donkey anti-mouse Alexa Fluor 488 antibody (715-545-150, 1:500, Jackson ImmunoResearch, West Grove, PA, USA). After a brief wash, samples were stained with NucBlue (R37605, Themo Fisher Scientific, Waltham, MA, USA) for nuclei detection. Fluorescent images were acquired using Nikon C1si laser scanning confocal microscope abb. LSCM (Nikon, Tokyo, Japan) and EVOS FL (AMG, Bothell, WA, USA).
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