Third instar wandering larvae were dissected in 1 × Ca2+ free saline (0.128 M NaCl, 2 mM KCl, 5 mM EGTA, 4 mM MgCl2, 5 mM HEPES, 0.0355 M Sucrose) at room temperature (22°C) on a sylgard coated dish. Larvae were incubated in 4% formaldehyde in PBS for 20 min at room temperature, and permeabilized overnight at 4°C (PBS + 0.3% Tween). Samples were immunostained with rabbit anti-GFP at 1:500 (A-11122, Invitrogen) overnight, then Alexa488-conjugated-donkey anti-rabbit IgG at 1:500 (711-545-152, Jackson ImmunoResearch Laboratories) and Alexa647-conjugated-goat anti-HRP at 1:100 (123-605-021, Jackson ImmunoResearch Laboratories) overnight. Samples were imaged with a 20 × /N.A.0.60 oil objective on a Leica SPE laser scanning confocal microscope (JH Technologies), with identical imaging parameters among different genotypes. Images were processed with ImageJ using only linear adjustments of contrast and color.
Alexa488 conjugated donkey anti rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa488-conjugated donkey anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is used to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
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Lab products found in correlation
Cy3 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (2 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Alexa 647 conjugated goat anti hrp, by Jackson ImmunoResearch (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Biotin conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Cy3 strepavidin, by Jackson ImmunoResearch (1 mentions)
Rabbit anti c ebpβ, by Santa Cruz Biotechnology (1 mentions)
Mouse anti pax7, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti neun antibody, by Merck Group (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Rabbit polyclonal anti nrg1, by Santa Cruz Biotechnology (1 mentions)
4 protocols using alexa488 conjugated donkey anti rabbit igg
1
Larval Dissection and Immunostaining
2
In situ TUNEL and Immunohistochemistry for Muscle
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In situ TUNEL assays were performed according to the manufacturer's instructions (Roche, Laval, QC, Canada) and counter-stained with DAPI (0.5 μg/ml) for 5 min. For immunohistochemistry, muscle sections were air-dried 30 min at 65 °C and fixed 10 min in 4% paraformaldehyde. After washes, antigen retrieval was done for 20 min at 92 °C with citrate buffer (10 mM citric acid, 0.05% Tween-20, pH 6.0) and sections were cooled to 20 °C. Sections were permeabilized 10 min in 0.5% Triton X-100, washed and blocked 1 h in 5% normal donkey serum (Jackson Immunoresearch, West Grove, PA, USA). Antibodies used for detection were: mouse anti-Pax7 (DSHB, 1/100), and rabbit anti-C/EBPβ (Santa Cruz Biotechnology, Dallas, TX, USA, SC-150, 1/100) biotin-conjugated donkey anti-mouse IgG (Jackson Immunoresearch) with Cy3-strepavidin (Jackson Immunoresearch), and Alexa488-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch). All primary antibodies were added on sections and incubated at 4 °C overnight. Secondary antibody labeling was done for 1 h.
3
Immunofluorescent Visualization of Neuronal Markers
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Monkey LX was deeply anaesthetized with pentobarbital sodium (50 mg kg−1, intravenously) and perfused transcardially with 0.1 M PBS, followed by 10% formalin in PBS. The brain was removed from the skull, blocked into pieces and equilibrated with 30% sucrose in PBS. Frozen sections were cut at 50 μm thickness in the coronal plane. To visualize the immunoreactive signals of ChR2-EYFP and NeuN, the sections were immersed in 1% skim milk for 1 h and incubated overnight with rabbit anti-green fluorescent protein antibody (1:1,000 dilution, Life Technologies) and mouse monoclonal anti-NeuN antibody (1:1,000 dilution, Millipore) in PBS containing 1% normal donkey serum. The sections were then incubated in the same fresh medium containing Alexa488-conjugated donkey anti-rabbit IgG (1:400 dilution, Jackson ImmunoResearch) and Cy3-conjugated donkey anti-mouse IgG (1:400 dilution, Jackson ImmunoResearch). Images of the sections were digitally captured using an optical microscope equipped with a high-grade charge-coupled device camera (Biorevo, Keyence).
4
Immunodetection of NRG1 in AdMSCs
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For immunocytochemistry, AdMSCs and NRG1-AdMSCs were grown in four-well Lab-Tek II chamber slides (Nunc, USA) at a density of 2×104 cells/mL. Briefly, the cells were fixed with 4% paraformaldehyde for 30 min at 4°C. The cells were incubated in blocking solution (10% normal donkey serum) for 30 min and then with rabbit polyclonal anti-NRG1 (1:200; Santa Cruz Biotechnology, USA) for 16 h at 4°C. After washing with PBS, the cells were incubated with Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch, USA) for 1 h at room temperature (22 ± 3°C). After final washing with PBS, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
For immunohistochemical staining, the 40-μm-thick coronal sections were incubated with 10% normal donkey serum for 1 h and then with the following primary antibodies for 16 h at 4°C: rabbit polyclonal anti-NRG1 (1:200; Santa Cruz Biotechnology), mouse monoclonal anti-human nuclei (1:200; Millipore Corporation, USA). After washing with PBS, the sections were incubated with the following secondary antibodies for 2 h at room temperature (22 ± 3°C): Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch), Alexa488-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch). Then, after washing with PBS, the sections were mounted and observed using a confocal microscopy (LSM 510 Meta; Carl Zeiss Co., Germany).
For immunohistochemical staining, the 40-μm-thick coronal sections were incubated with 10% normal donkey serum for 1 h and then with the following primary antibodies for 16 h at 4°C: rabbit polyclonal anti-NRG1 (1:200; Santa Cruz Biotechnology), mouse monoclonal anti-human nuclei (1:200; Millipore Corporation, USA). After washing with PBS, the sections were incubated with the following secondary antibodies for 2 h at room temperature (22 ± 3°C): Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch), Alexa488-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch). Then, after washing with PBS, the sections were mounted and observed using a confocal microscopy (LSM 510 Meta; Carl Zeiss Co., Germany).
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