The animals were anesthetized, transcardially perfused, and post-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at 72 h after TBI. Brain tissues were embedded in paraffin and serial sections were cut at 5 µm intervals from bregma -2.0 mm to bregma -7.0 mm to collect the entire lesioned cortex and mounted on slides. For immunohistochemistry, slides were incubated with primary antibody against glial fibrillary acidic protein (GFAP) (goat polyclonal to GFAP, 1:100, Abcam, New Territories, HK), Ionized calcium binding adaptor molecule 1 (Iba-1) (goat polyclonal to Iba1, 1:100, Abcam), myeloperoxidase (MPO) (rabbit polyclonal to MPO, 1:100, Abcam), and CD3 (rabbit polyclonal to CD3, 1:100, Abcam) at 4°C overnight. Following primary antibody incubation, slides were incubated with secondary antibodies (Alexa 488 donkey-anti-goat IgG, Invitrogen, 1:300; Alexa 594 conjugated goat-anti-rabbit IgG, 1:300) for 2 h, and the negative control was secondary antibody only). All the sections were observed using a fluorescence microscope (Olympus BX51, DP71), and the number of positive cells near the injured areas was counted (8 to 10 sections per brain, 500 µm apart) in a blinded manner.
Alexa 488 donkey anti goat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor® 488 donkey anti-goat IgG is a secondary antibody conjugate used for the detection of goat primary antibodies in immunoassays. It is designed to bind to the Fc region of goat IgG, allowing for the visualization of target proteins or cellular structures labeled with a goat primary antibody. The Alexa Fluor® 488 dye provides bright green fluorescent labeling of the target.
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Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Alexa 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fv10i confocal microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole dapi for nuclear staining, by Merck Group (1 mentions)
Freezing microtome, by Leica (1 mentions)
Agonists and antagonists, by Bio-Techne (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Anti ve cadherin antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Anti cd31 antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti vinculin, by Merck Group (1 mentions)
Rat anti l1 cam, by Merck Group (1 mentions)
Goat anti chat, by Merck Group (1 mentions)
Goat anti brn3a, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 647, by BioLegend (1 mentions)
Apc efluor 780, by BioLegend (1 mentions)
Brilliant violet 421, by BioLegend (1 mentions)
Live dead fixable yellow dead cell stain, by Thermo Fisher Scientific (1 mentions)
13 protocols using alexa 488 donkey anti goat igg
1
Immunohistochemical Analysis of TBI-Induced Neuroinflammation
2
Visualization of Neuronal Marker Expression
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After fixing with 4% paraformaldehyde for 15 min at 37 °C, mice neuronal cultures were washed with phosphate-buffered saline (PBS) and permeabilized with 0.2% Triton X-100, followed by incubation of primary antibodies overnight at 4 °C. The primary antibodies were diluted as follows: Homer 1a (1:50) and MAP-2 (1:200). Then, cells were incubated with secondary antibodies (Alexa 488 donkey-anti-goat IgG (Invitrogen), 1:300; Alexa-594-conjugated goat-anti-mouse IgG, 1:300) for 2 h. Cultures were dehydrated with ethanol and mounted with 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining (Sigma). Images were captured using an Olympus FV10i Confocal Microscope (Olympus, Tokyo, Japan). All images from one experiment were acquired using the same exposure time to allow comparisons of relative levels of immunoreactivity between the different treatment conditions. At least six images of each group were taken by an evaluator blinded to the experimental conditions.
3
Immunohistochemical Analysis of Dopaminergic Neurons
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To detect TH-positive DA neurons, serial 30 µm thick coronal sections were cut on a freezing microtome (Leica, Nussloch, Germany). The brain tissue sections were pre-treated with 1% hydrogen peroxide for 15 min and incubated with a rabbit anti-TH antibody (1∶200) overnight at 25°C in the presence of 0.3% Triton X-100 and normal goat serum. The peroxidase activity was visualized by incubating the sections with DAB in 0.05 M tris-buffered saline (pH = 7.6). After several rinses with PBS, the samples were mounted on gelatin-coated slices, dehydrated, and coverslipped in histomount medium. For the Iba-1 immunofluorescence staining, the brain tissue sections were incubated with a rabbit anti-Iba-1 antibody (1∶200), and Alexa 488 donkey-anti-goat IgG (Invitrogen, 1∶300) was used as the secondary antibody for 2 h of incubation. The results were evaluated independently by two specialized neuropathologists who were blinded to all of the experimental groups. The number of TH-positive cells was counted in SNpc at 40×magnification, and the optical density of TH-positive fibers in the striatum (ST) was measured at 100×magnification.
4
Brain Tissue Immunostaining Protocol
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Cells transduced with C or sh2612 were identified in 35 μm brain sections by immunohistofluorescence using a goat polyclonal antibody against eGFP (1:2000, AbCam, Cambridge, MA; Supplementary Table 3 ). Following an overnight incubation with this antibody, the sections were incubated for 1 h at room temperature with an Alexa 488 donkey antigoat IgG (1:500, Invitrogen), followed by 1 min incubation with Hoechst 33258 reagent (Thermo Fisher; 1:10,000) to stain cell nuclei.
5
Verification of Viral Infection in Hypothalamic ARC
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To verify the location of microinjections targeting GFP-expressing lentiviruses to the ARC of the hypothalamus, we perfusion-fixed the brain of the injected rats 22–25 days after the initial infection with 4% paraformaldehyde-PBS pH 7.4, blocked the portion of the brain containing the hypothalamus, and serially sectioned the blocks at 30 μm intervals. The sections were then incubated overnight at 4 °C with goat polyclonal antibodies against GFP (Abcam, Cambridge, MA; 1:2,000 dilution), and the reaction was developed the next day to a green colour using Alexa 488 donkey antigoat IgG (Invitrogen, 1:500). Fluorescent images were acquired with an AxioImager A2 Zeiss fluorescent microscope.
6
Antibody Reagents for Neurobiological Signaling
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Primary antibody against Homer 1a was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phosphor-ERK1/2 (p-ERK) and total ERK1/2 (ERK) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody against β-actin was obtained from Sigma (St Louis, MO, USA). The secondary antibodies for immunoblotting were horseradish peroxidase-conjugated anti-rabbit and anti-goat IgG (Santa Cruz Biotechnology). The secondary antibodies for immunostaining were Alexa 488 donkey anti-goat IgG and Alex 594 donkey anti-rabbit IgG) (Invitrogen, Carlsbad, CA, USA). Agonists and antagonists were obtained from Tocris Bioscience (Bristol, UK).
7
Immunofluorescence Staining of Endothelial Cells
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Attached cells were briefly fixed with ice-cold acetone and blocked in antibody dilution buffer (2 % BSA/PBS) for 1 hour at room temperature. After removal of the blocking solution, primary antibodies anti-VE-cadherin antibody (1:100; Santa Cruz, Dallas, TX, USA) and anti-CD31 antibody (1:100; Santa Cruz) were added and cells were incubated at 4 °C overnight. The cells were then washed and incubated with Alexa 488 donkey anti-goat IgG and Alexa 488 goat anti rabbit IgG (Invitrogen, Carlsbad, CA, USA) for 30 minutes at room temperature. Immunofluorescence was observed under a fluorescent microscope (Olympus).
8
Histological Examination of Brain Tissues
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The animals were transcardially perfused and postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer. Brain tissues were embedded in paraffin. Serial sections were cut at 5 μm on a cryostat and mounted on the slides. For hematoxylin and eosin (H&E) staining, deparaffinized sections were stained with H&E according to the routine protocol. For immunostaining, deparaffinized sections were boiled in unmasking antigen buffer (10 mM sodium citrate buffer, pH 6.0) and incubated in 3% hydrogen peroxide for 10 min. After treating with blocking buffer for 1 h at room temperature, diluted primary antibody was added and incubated overnight at 4 °C. For DAB staining, sections were incubated with secondary antibodies (HRP-conjugated IgG) for 30 min. DAB dilution was added on sections and the reaction was stopped according to appropriate staining intensity. Sections were counterstained in hematoxylin. For double-labeled immunofluorescence staining, sections were incubated with secondary antibodies (Alexa 488 donkey-anti-goat IgG (Invitrogen), 1:300; Alexa-594-conjugated goat-anti-mouse IgG, 1:300) for 2 h. After staining, sections were coversliped with different mounting mediums. All the sections were observed using a fluorescence microscope (Olympus BX51, DP71).
9
Antibody Labeling for Immunofluorescence and Western Blotting
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The following antibodies were used in this study: rabbit anti‐MYO9B (#12432‐1‐AP, Proteintech), mouse antivinculin (#05‐386, Millipore), rat anti‐myelin basic protein (MBP; #MAB386, Millipore), rat anti‐L1‐CAM (#MAB5272, Millipore), chicken anti‐NF‐M (#822701, Biolegend), goat anti‐ChAT (#AB144P, Millipore), and goat anti‐Brn3a (SC:8429, Santa Cruz Biotechnology).
For immunofluorescence, secondary antibodies included fluorescein (FITC)‐ and rhodamine (TRITC)‐conjugated donkey anti‐rat, chicken, or rabbit IgG (Jackson ImmunoResearch), and Alexa‐488 donkey anti‐Goat IgG (#A11055, Invitrogen).
For Western blotting, secondary antibodies included horseradish peroxidase‐conjugated goat antirabbit (#P0448, DAKO) and rabbit antimouse (#P0260, DAKO) immunoglobulins.
For immunofluorescence, secondary antibodies included fluorescein (FITC)‐ and rhodamine (TRITC)‐conjugated donkey anti‐rat, chicken, or rabbit IgG (Jackson ImmunoResearch), and Alexa‐488 donkey anti‐Goat IgG (#A11055, Invitrogen).
For Western blotting, secondary antibodies included horseradish peroxidase‐conjugated goat antirabbit (#P0448, DAKO) and rabbit antimouse (#P0260, DAKO) immunoglobulins.
10
Multicolor Flow Cytometry Immunophenotyping
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The following reagents were purchased from BD Bioscience, eBioscience or Biolegend: biotinylated–αCD11c and αSca-1; FITC–conjugated αI-A/I-E; Alexa Fluor 488–αCD3, αCD4, αCD8, αCD11b, αCD19, and αB220; PE–αCD11c, αCD115, αCD172α (SIRPα), αCD317 (PDCA-1); PerCP5.5–conjugated αTCRβ; PerCP-eFluor 710–αCD135; PE-Cy7–conjugated αCD11c, αCD86; Alexa Fluor 647–αBcl6 and mouse IgG1 isotype control; APC-eFluor 780–αCD117 (c-Kit); Brilliant Violet 421–αCD11c, αB220 and αI-A/I-E; and Cytoperm/Cytofix solution and Perm/Wash buffer. FITC–αKi-67 was from Abcam. Streptavidin-Alexa Fluor 555, Alexa488–Donkey anti-goat IgG, Prolong Gold antifade mounting medium, and LIVE/DEAD Fixable Yellow Dead cell stain were purchased from Invitrogen.
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