Mice were anesthetized and perfused by 4% PFA transcardially, and the dissected brains were postfixed with 4% PFA overnight. After immersion in 30% sucrose in PBS, 30-μm-thick coronal sections were cut on a cryostat microtome. For BrdU immunohistochemistry, sections were treated first with 2 M HCl at 37 °C for 15 min, and then blocked in 2% BSA (Sigma-Aldrich) in DPBS supplemented with 1% Triton-X 100 (Sigma-Aldrich) at 4 °C overnight. The sections were then incubated with primary antibodies at 4 °C overnight. The following antibodies were used: rat anti-BrdU (1:200; Serotec), goat anti-doublecortin (1:50; Santa Cruz), and mouse anti-NeuN (1:200; Millipore). Fluorophore-conjugated secondary antibodies were used at a dilution of 1:1000 and incubated with the sections for 1.5 h at RT. Secondary antibodies used were (all from Thermo Fisher Scientific) Alexa 594-conjugated donkey anti-rat, Alexa 488-conjugated donkey anti-goat, Alexa 488-conjugated donkey anti-rat, and Alexa 594-conjugated donkey anti-mouse. Cell nuclei were visualized by DAPI (Sigma-Aldrich).
Alexa488 conjugated donkey anti goat
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa488-conjugated donkey anti-goat is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and bind to goat primary antibodies in immunoassays and other applications requiring fluorescent labeling.
Automatically generated - may contain errors
Lab products found in correlation
Formalin, by Merck Group (2 mentions)
Anti rabbit igg h l antibodies, by Thermo Fisher Scientific (2 mentions)
Dapi staining solution, by Merck Group (2 mentions)
Dc250, by Leica (2 mentions)
Rat anti brdu, by Bio-Rad (1 mentions)
Goat anti doublecortin, by Santa Cruz Biotechnology (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Iba 1, by Abcam (1 mentions)
Lcx100 imaging system, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated donkey anti goat, by Jackson ImmunoResearch (1 mentions)
Rabbit anti β gal, by MP Biomedicals (1 mentions)
Goat anti prox1, by R&D Systems (1 mentions)
Goat anti vegfr3, by R&D Systems (1 mentions)
Cy5 conjugated donkey anti rat, by Jackson ImmunoResearch (1 mentions)
8 protocols using alexa488 conjugated donkey anti goat
1
BrdU Immunohistochemistry of Adult Neurogenesis
2
Immunofluorescence Assay of Iba1 and P2Y12 in Heart Tissue
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Hearts were sectioned into approx. 7-µm slices for immunofluorescence assays using a freezing microtome. The sections were incubated with anti-ionized calcium binding adapter molecule 1 (Iba1) (1 : 100; Abcam, Cambridge, UK) and anti-P2Y12 (1 : 50, Novus, Centennial, USA) as primary antibodies diluted in phosphate-buffered saline (PBS) overnight at 4°C. After washing 3 times with PBS, the sections were incubated with Alexa 546-conjugated donkey anti-rabbit (1 : 200; Thermo Fisher Scientific, Waltham, USA) and Alexa 488-conjugated donkey antigoat (1 : 200, Thermo Fisher Scientific) antibodies for 2 h. The sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Life Technologies, Grand Island, USA) to identify nuclei. Olympus LCX100 Imaging System (Olympus Corp., Tokyo, Japan) and ImageJ software (National Institutes of Health, Bethesda, USA) were used for image acquisition and analysis.
3
Immunofluorescence Analysis of Lymphatic Markers
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The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam). The following secondary antibodies were used: Alexa 488-conjugated donkey anti-rabbit (Molecular Probes), Alexa 488-conjugated donkey anti-guinea pig (Molecular Probes), Alexa 488-conjugated goat anti-hamster (Molecular Probes), Alexa 488-conjugated donkey anti-goat (Molecular Probes), Cy3-conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories), Cy3-conjugated donkey anti-goat (Jackson ImmunoResearch Laboratories), and Cy5-conjugated donkey anti-rat (Jackson ImmunoResearch Laboratories).
4
Immunohistochemical Analysis of Glial Activation
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Cryosections were washed three times with PB and blocked in 10% normal donkey serum in PB for 1 h at room temperature. Sections were then incubated with goat anti-OPN polyclonal antibody (1:1000), rabbit polyclonal anti-ionized calcium binding adaptor molecule 1 (Iba1) antibody (1:1000; Wako Pure Chemical Industries, Osaka, Japan), or rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:1000; Chemicon, Temecula, CA, USA) in PB for 5 h at room temperature. Sections were subsequently washed in PB and incubated with Alexa 488-conjugated donkey anti-goat (1:1000; Molecular Probes, Eugene, OR, USA) or Cy3-conjugated anti-rabbit IgG (1:1000) for 2 h. Cell nuclei were counterstained with DAPI. Fluorescent specimens were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA) and imaged using a confocal microscope (Carl Zeiss Co., Ltd., Jena, Germany).
5
Immunofluorescent Detection of Vpr in Daudi Cells
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Daudi cells were adsorbed on polyLysine-treated slides and fixed with Formalin (Sigma) containing 1% Triton-X-100. Cells were labeled with anti-Vpr antibody followed by labeling with a mixture of Alexa488-conjugated donkey anti-goat and anti-rabbit IgG (H + L) antibodies (Life Technologies). Nuclei were stained with DAPI staining solution from Sigma Aldrich. Cells were mounted on glass slides covered with anti-fade medium (Hardset Vectashield). Two-color images were obtained with a light microscope, Leica DC250 (Leica) with a Plan Apo 63 ×/1.32–0.6 oil-immersion objective lens. Digital images were processed with the ImageJ software (NIH Image). Percentages of Vpr+ Daudi B-cells were determined by using the Cell Counting plugin of ImageJ. A minimum of 750 cells were counted per 6-well plate.
6
Imaging of α2-Macroglobulin Binding on Malaria IEs
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HB3VAR06+ IEs (1.2 mL, 5% hematocrit) were pelleted, washed (3×, PBS with 1% Ig-free BSA), and incubated with α2M (500 μL, 100 nM, room temperature, 30 min). Following additional washing, the IEs were incubated with polyclonal goat-anti- α2M (500 μL, 1:3,000) then Alexa 488-conjugated donkey-anti-goat (Life Technologies, 1:10,000) and DAPI (1 μg/mL). Micrographs of live, unfixed IEs were obtained using a Nikon TE 2000-E confocal microscope equipped with a 60× oil immersion lens (N.A. 1.4) and Nikon EZ-C1 3.5 software (Nikon Instruments, Amsterdam, Netherlands). Images were analyzed using Image J64 software (http://imagej.nih.gov/ij/ ) [66 (link)].
7
Immunohistochemistry Markers for Vascular Imaging
8
Daudi B-Cell Vpr Localization Analysis
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Daudi cells were adsorbed on polyLysine-treated slides and xed with Formalin (Sigma) containing 1% Triton-X-100. Cells were labeled with anti-Vpr antibody followed by labeling with a mixture of Alexa488conjugated donkey anti-goat and anti-rabbit IgG (H+L) antibodies (Life Technologies). Nuclei were stained with DAPI staining solution from Sigma Aldrich. Cells were mounted on glass slides covered with anti-fade medium (Hardset Vectashield). Two-color images were obtained with a light microscope, Leica DC250 (Leica) with a Plan Apo 63 × /1.32-0.6 oil-immersion objective lens. Digital images were processed with the ImageJ software (NIH Image). Percentages of Vpr + Daudi B-cells were determined by using the Cell Counting plugin of ImageJ. A minimum of 750 cells were counted per 6-well plate.
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