After treatments, the cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 m. They were then permeabilized with 0.25% Triton X-100 for 10 m, blocked with 5% bovine serum albumin for 30 m, incubated with primary antibody mouse monoclonal anti-TOM20 (BD, USA) and rabbit monoclonal anti-phospho-Drp1S616 (Cell Signaling) overnight at 4°C. Then, they were washed and incubated with secondary antibody (Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG) and Alexa Fluor 594 goat anti-mouse IgG (H + L) antibody; Molecular Probes) for 1 h at 37°C. Further, they were washed with phosphate-buffered saline. The nuclei were stained with DAPI (Invitrogen). Images were visualized under a 100× objective Olympus FluoView1000 confocal microscope using anti-p-drp1 (s616) (1:1000 dilution, green) and anti-Tom20 (a marker of mitochondria; 1:1000 dilution, red) antibodies. The images were quantified with the ImageJ software.
Alexa fluor 488 donkey anti rabbit immunoglobulin g igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG) is a secondary antibody conjugate designed for use in immunofluorescence applications. It is composed of donkey-derived anti-rabbit IgG antibodies labeled with Alexa Fluor 488 dye, which emits green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti tom20, by BD (1 mentions)
Mvh antibody, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Rabbit anti collagen type 1, by Merck Group (1 mentions)
Alexa fluor 546 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti s100, by Abcam (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Anti cd68, by Santa Cruz Biotechnology (1 mentions)
Anti neutrophil elastase, by Santa Cruz Biotechnology (1 mentions)
Anti adamts 1, by Abcam (1 mentions)
Alexa fluor 555 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by Leica (1 mentions)
7 protocols using alexa fluor 488 donkey anti rabbit immunoglobulin g igg
1
Visualizing Mitochondrial Dynamics
2
Indirect Immunolocalization of H3 Phosphorylation
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Indirect immunolocalization was performed as described previously (He and Davie, 2006 (link)), using rabbit polyclonal antibodies against H3S10ph (1:250 dilution; Santa Cruz Biotechnology, Dallas, TX) and rat monoclonal antibodies against H3S28ph (1:250 dilution; Sigma-Aldrich). Peptide Competition assay was done by Standard Novus protocol (www.novusbio.com/support/support-by-application/peptide-competition/protocol.html ) using H3 peptides from Abcam (human histone H3 (phospho S10) peptide [ab11477]; human histone H3 (phospho S28) peptide [ab14793; Supplemental Figure S9). Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG; Molecular Probes, Eugene, OR) and Texas red– or AMCA-conjugated donkey anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Control experiments including epitope peptide blocking or primary antibody omission demonstrated the specificity of the antibodies used.
3
Immunostaining of Mouse Testicular Cells
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Squash samples were prepared as previously described46 (link). The testes of adult C57Bl/6 mice were dissected and fixed in freshly prepared 2% formaldehyde in PBS (phosphate-buffered saline) (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.7 mM KH2PO4 pH 7.4) containing 0.05% Triton X-100 (Sigma) and 5 mM sodium orthovanadate for 5–10 min at room temperature. During this time, seminiferous tubules were liberated and dispersed in the fixative. Pieces of tubules were placed on a slide and gently minced with tweezers, and then a coverslip was added and the cells were squashed by exerting pressure on the coverslip. The slides were frozen in liquid nitrogen and the coverslips were removed. The slides were immediately placed in PBS for three subsequent 5 min rinses and blocked 1 h in 5% BSA in PBS. The primary antibody incubation was carried out at 4 °C in 1% BSA solution with CaMKIV antibody (Abcam) and MVH antibody (Abcam). Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG) (Molecular Probes) was used as secondary antibody. The nuclei were stained by Hoechst 33342 (Sigma) and the slides were digitally imaged using a fluorescence microscope (Nikon, T80i, Japan).
4
Antibody Validation for Cell Signaling Studies
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Commercially available antibodies used for IP, immunoblotting, and immunofluorescence were as follows: Goat anti–apolipoprotein B (EMD, Hayward, CA; #178467 1:500 for immunoblot); rabbit anti-FABP5 human (Proteintech, Rosemont, IL; #12348-1-AP 1:2000 for immunoblot); rat anti-FABP5 monoclonal antibody (Thermo Fisher Scientific, Waltham, MA; ma5-24029 1:300 for immunofluorescence); rabbit anti–PC1 LF-41 was a gift from L. Fisher (National Institute of Dental and Craniofacial Research, Bethesda, MD [Fisher et al., 1989 , 1995 (link); Bernstein et al., 1996 (link)]; 1:5000 for immunoblot); rabbit anti-collagen type I (Millipore, Hayward, CA; #AB745 1:300 for immunofluorescence); Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG) (Invitrogen, Carlsbad, CA; #A-21206 1:250 for immunofluorescence); Alexa Fluor 546 goat anti-rat IgG (Invitrogen #A-11081 1:250 for immunofluorescence).
5
Validating Optogenetic Transduction in Schwann Cells
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Prior to examining the effect of optogenetics on SCs, the efficiency of transfected SCs was tested. Coverslips seeded with SCs (2 × 104 cells) were plated into a 24-well plate. At DIV 7 after CatCh transfection, SC samples were fixed with 4% paraformaldehyde (PFA) and immunostained with anti-green fluorescent protein (GFP) (1:1,000; Abcam) and anti-S100ß (1:300; Abcam), followed by the secondary antibodies Alexa Fluor® 488 donkey anti-rabbit immunoglobulin G (IgG, 1:1,000; Invitrogen) and Alexa Fluor® 568 goat anti-mouse IgG (1:500, Invitrogen). The GFP protein used herein shares approximately 97.8% sequence identity with YFP protein. SC transfection was confirmed by calculating the number of GFP protein-stained SCs. Samples were then counterstained with DAPI (Life Technologies). The number of GFP+-S100ß+ SCs was calculated from multiple fields of view in confocal fluorescence images and quantified. The total number of S100ß+ SCs stained with DAPI was first calculated, and the number of GFP+ cells in S100ß+ SCs was confirmed (n = 3, five random regions).
6
Immunohistochemical Analysis of NeuroD1 in Tissue
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Slide-mounted sections (20 μm) were washed in 0.1M PBS (pH 7.4) and then incubated in immunoblocking buffer (2.5% bovine serum albumin [BSA], 0.1% Triton X-100 in PBS) for 1 h at room temperature. Subsequently, sections were incubated with Rabbit anti-NeuroD1 primary antibody (1:250, Abcam, Cambridge, UK). After overnight incubation at 4 °C, the sections were washed in PBS and incubated for 3 h with fluorescent secondary antibody Alexa Fluor 488 Donkey anti-rabbit immunoglobulin G (IgG) (1:400, Invitrogen, MOLECULAR PROBES®, Eugene, Oregon, USA). The nuclear dye DAPI (4′,6-diamidino-2-phenylindole; Sigma–Aldrich, Buchs, Switzerland) was added to secondary antibodies solution for nuclear staining. Sections were then rinsed in PBS and coverslipped with fluorescent mounting medium.
7
Dual Immunofluorescence Assay for ADAMTS1, CD68, and Neutrophil Elastase
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For the dual immunofluorescence assay, frozen sections were incubated with anti-ADAMTS1 (Abcam, Cambridge, MA, USA), anti-CD68, or anti-neutrophil elastase (Santa Cruz, CA, USA) primary antibody and then Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG) or Alexa Fluor 555 donkey anti-goat IgG (Invitrogen, Carlsbad, CA, USA) secondary antibody. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Negative controls replaced primary antibody with IgG. The fluorescence signal was monitored by confocal laser scanning microscopy (Leica, Wetzlar, Germany).
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