Neurospheres treated with DMSO or PTC-209 were dissociated into single cells, and were cyto-spun to attach on glass-slides. Cells were then fixed with 4% paraformaldehyde (PFA), washed with PBS, blocked for non-specific binding using blocking solution (PBS supplemented with 5% donkey serum and 0.3% Triton X-100) and incubated overnight with the indicated primary antibodies [anti-GFAP (Z0334) and anti-Vimentin (M7020), DAKO; anti-Nestin (MAB5326, Millipore)]. After PBS wash the next day, the cells were incubated with secondary antibodies (Alexa-Fluor-594 conjugated Donkey anti-Rabbit (#711-585-152) and Alexa-Fluor-488 conjugated Donkey anti-Mouse (#715-545-150); Jackson ImmunoResearch) for 1 hour, and washed with PBS. Finally, the slides were embedded with Mounting Media with DAPI (Vector Laboratories, USA). Images were captured at 60X oil objective in confocal microscope (Nikon, USA).
Alexa fluor 488 conjugated donkey anti mouse
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 488-conjugated donkey anti-mouse is a secondary antibody product that is conjugated with the fluorescent dye Alexa Fluor 488. It is designed to detect and bind to mouse primary antibodies, enabling visualization and detection in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 conjugated donkey anti mouse, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 594 conjugated donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Mounting media with dapi, by Vector Laboratories (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Extravidin cy3, by Merck Group (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Alexa fluor 594 conjugated donkey anti guinea pig, by Jackson ImmunoResearch (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ds qi1mc camera, by Nikon (1 mentions)
A83 01, by Bio-Techne (1 mentions)
Pd173074, by Merck Group (1 mentions)
Mounting medium, by Merck Group (1 mentions)
Cy3 conjugated donkey anti goat, by Jackson ImmunoResearch (1 mentions)
Goat polyclonal anti iba1, by Abcam (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Mouse monoclonal anti tlr4, by Abcam (1 mentions)
Tcs sp2, by Leica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti ve cadherin, by BD (1 mentions)
11 protocols using alexa fluor 488 conjugated donkey anti mouse
1
Characterization of Neural Progenitor Cells
2
Immunofluorescent Labeling of Brain Slices
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After recordings, brain slices were fixed overnight by immersion in 4% formaldehyde in phosphate buffered saline (PBS) at 4 °C. Next, the slices were washed 3 × 10 min in PBS and incubated in PBS solution containing 10% normal donkey serum (NDS) and 0.6% Triton X-100 (for blocking unspecific binding sites and permeabilization, respectively) for 3 h at room temperature. Afterwards, the slices were washed 1 × 10 min in PBS and incubated in PBS solution containing primary antibodies against TH (mouse anti-TH (F-11), 1:250, Santa Cruz Biotechnology, Cat# sc-25269, Lot# G1318, RRID:AB_628422 ), ExtrAvidin-Cy3 (biocytin binding protein, 1:200, Sigma-Aldrich, Cat# E4142, Lot# SLBT2189), 2% NDS and 0.3% Triton X-100 for 48–72 h at 4 °C. The slices were then washed 4 × 10 min in PBS and incubated in PBS solution containing 2% NDS and Alexa Fluor 647-conjugated donkey anti-mouse (1:400, Jackson ImmunoResearch Labs, Cat# 715-606-151, Lot# 138538, RRID:AB_2340866 ) or Alexa Fluor 488-conjugated donkey anti-mouse (1:400, Jackson ImmunoResearch Labs, Cat# 715-545-151, Lot# 127820, RRID:AB_2341099 ) secondary antibody for 24 h at 4 °C. After subsequent washing in PBS (4 × 10 min), the slices were mounted onto glass slides and coverslipped with aqueous mounting medium (Fluoroshield with DAPI, Sigma-Aldrich, Cat# F6057, Lot# SLCC1782, SLBX6915 or SLCC1782).
3
Immunohistochemical Analysis of Pancreatic Beta Cells
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Pancreas samples were fixed in 4% paraformaldehyde (MilliporeSigma) and embedded in paraffin wax. Sections (5 μm) were rehydrated followed by antigen retrieval with 2N hydrochloric acid for 20 minutes at 37°C and subsequent 0.05% trypsin solution (MilliporeSigma) for 15 minutes at 37°C. Sections were incubated in blocking buffer (0.25% BSA, 0.25% Triton X-100 in PBS) before incubation with both a mouse monoclonal anti-BrdU antibody (1:100, MilliporeSigma, B8434) and guinea pig polyclonal anti-insulin antibody (1:200, Dako, A0564) for 2 hours at 37°C.
Alexa Fluor 488–conjugated donkey antimouse (1:100, Jackson ImmunoResearch) and Alexa Fluor 594–conjugated donkey anti–guinea pig (1:100, Jackson ImmunoResearch) were used to visualize staining. A Nikon Eclipse TE2000-U microscope was used for acquiring images at original magnification ×200 using a Nikon DS-Qi1MC camera.
Sections were analyzed from across the pancreas for each animal, and all islets on each section were analyzed. ImageJ image analysis software (NIH) was used to count the number of BrdU+ β cells, total number of β cells, and cross-sectional area for each islet.
Alexa Fluor 488–conjugated donkey antimouse (1:100, Jackson ImmunoResearch) and Alexa Fluor 594–conjugated donkey anti–guinea pig (1:100, Jackson ImmunoResearch) were used to visualize staining. A Nikon Eclipse TE2000-U microscope was used for acquiring images at original magnification ×200 using a Nikon DS-Qi1MC camera.
Sections were analyzed from across the pancreas for each animal, and all islets on each section were analyzed. ImageJ image analysis software (NIH) was used to count the number of BrdU+ β cells, total number of β cells, and cross-sectional area for each islet.
4
Efficient Trophoblast Differentiation from hiPSCs
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BMP-4 is known to trigger the differentiation of human induced pluripotent stem cells (hiPSCs) into trophoblast-like cells [16 (link)]. HiPSCs were cultured in our own in-house produced Essential 8 medium (E8). The trophoblast differentiation protocol was modified from Amita [17 (link)]. Briefly, cells were plated at low density (4 × 103 cells per cm2) in Matrigel-coated cell culture plates in E8 medium. After 48 h, the E8 medium was replaced with differentiation medium containing 1 μM A8301 (Tocris, UK) and 0.1 μM PD173074 (Sigma, US), as well as 10 ng/ml commercial BMP-4 (R&D Systems, USA) or the rhBMP-4 dimer produced in E. coli. In 48 h after the addition of the differentiation medium, the cells were fixed with 4% paraformaldehyde and immunostained for cdx2, a transcription factor known to be an early trophoblast marker. The primary antibody was an Anti-CDX2, clone CDX2-88 (BioGenex, US) and the secondary antibody was Alexa-Fluor-488 conjugated donkey-anti-mouse (Jackson ImmunoResearch, US). Counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI).
5
Double Immunofluorescence Analysis of TLR2, TLR4, and Iba1 in Rat Brain
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Two SD rats of the control group were randomly selected for double immunofluorescence analysis. The brain sections were blocked for 10 min in PBS containing 10% bovine serum albumin (Sigma, USA). The sections were then incubated overnight at 4°C with primary antibodies [1 : 100 rabbit polyclonal anti-TLR2 (OriGene-Antibody, USA), 1 : 100 mouse monoclonal anti-TLR4 (Abcam, USA), and 1 : 500 goat polyclonal anti-Iba1 (Abcam, USA)]. Further, the sections were incubated with secondary antibodies [1 : 800 Alexa Fluor 488-conjugated donkey anti-rabbit, 1 : 800 Alexa Fluor 488-conjugated donkey anti-mouse, and 1 : 800 Cy™ 3-conjugated donkey anti-goat (Jackson ImmunoResearch Lab. Inc., USA)] for 1 h at room temperature. Each of the abovementioned steps was followed by three 3-min rinses in 0.01% Tween 20/PBS. At the end of the procedure, the sections were covered with coverslip using a mounting medium (SIGMA, USA) containing DAPI to counterstain DNA in the nuclei and dried overnight. The confocal images were captured using a laser-scanning confocal microscope (Leica TCS SP2, Germany).
6
Colocalization of ChAT and NGF Markers
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To investigate the colocalization of the motor neuron marker ChAT with NGF, double staining of ChAT with NGF was performed, the sections were blocked in 5% normal donkey serum (Jackson ImmunoResearch) and incubated with polyclonal goat anti-ChAT antibody (1:150; Millipore, Bedford, MA, USA) at 4°C overnight, then reacted with Alexa-Fluor 594 conjugated donkey anti-goat (1:200; Jackson ImmunoResearch) at room temperature for 1 h. The sections were further incubated with monoclonal mouse anti-NGF antibody (1:200; Santa Cruz) at 4°C overnight, followed by incubation with Alexa-Fluor 488 conjugated donkey anti-mouse (1:200; Jackson ImmunoResearch). Finally, the sections were observed under a fluorescence microscope.
7
Antibody Panel for Endothelial Cell Analysis
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The following antibodies were used: anti-CD93 (R and D, AF1696, 1:500); anti-Erg (Abcam, Ab92513, 1:400); anti-Ki67 (Invitrogen, 14-5698-82, 1:200); anti-Podocalyxin (R and D, AF1556, 1:400); anti-CoupTFII (LSBio, LS-C356225, 1:200); anti-PECAM-1 (BD, 55370, 1:200 or Millipore, MAB1398z, 1:500); anti-Claudin-5 (Invitrogen, 352588, 1:200); anti-VE-cadherin (BD, 550548, 1:200); and anti-Cingulin (Invitrogen, 36–4401, 1:500). Alexa Fluor 488, 555 and 647 donkey secondary antibodies were from Jackson ImmunoResearch (1:400). Alexa Fluor 488 conjugated donkey anti-mouse was used to detect murine IgG (1:400, Jackson ImmunoResearch). The anti-uPAR antibody was kindly donated by Francesco Blasi, FIRC Institute of Molecular Oncology Foundation (IFOM), Milan, Italy (Tjwa et al., 2009 (link)).
8
Muscle Tissue Apoptosis and Inflammation
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Muscle samples were fixed in 4% paraformaldehyde at 4°C overnight. Then, they were conventionally prepared for paraffin embedding and sectioned into 4-μm slides. For the TUNEL assay, a One-Step TUNEL Assay Kit (Beyotime, Shanghai, China) was used. For immunofluorescence staining, tissue sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 minutes and then processed with 0.25% Triton-100 for 30 minutes. A primary antibody against NLRP3 (1 : 250) was purchased from Abcam (Cambridge, UK). A caspase-1 antibody (1 : 50) was purchased from Santa Cruz (TX, USA). The sections were incubated in primary antibodies at 4°C overnight. The secondary antibodies were Alexa Fluor 488-conjugated donkey anti-mouse and Alexa Fluor 594-conjugated donkey anti-rabbit antibodies and were purchased from Jackson ImmunoResearch (PA, USA). Then, the sections were stained with DAPI for 5 minutes in the dark, and micrographs were taken with a Leica DM2500 (Wetzlar, Germany) microscope.
9
Visualizing Yeast Microtubule Cytoskeleton
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Sporulating cells were harvested and fixed in 3.7% methanol-free formaldehyde for 15 minutes, washed twice with ddH2O and then suspended in 1 ml of SP buffer (1M sorbitol 10mM pH7.8 PBS). These cells were then spheroplasted at 37°C after adding 20μl 20T zymolyase at 5 mg/ml concentration and 1 μl β-mercaptoethanol; spheroplast formation was checked using a 100x phase objective on a Zeiss AxioMot2. Spheroplasted cells were washed with 1 ml SP buffer and resuspended in 500 μl of fresh SP buffer. Spheroplasted cells were then adhered to polylysine coated slides, blocked with blocking buffer (1% BSA, 0.1% Triton in PBS) and then rinsed three times with PBS.
Tubulin was detected using monoclonal mouse 12G10 anti-Tub1 antibody at 1:1000 concentration (Developmental Studies Hybridoma Bank). Cells were incubated with antibody for one hour, rinsed four times with PBS and then incubated with Cy2-conjugated donkey antimouse antibodies at 1:100 dilution for one hour (JacksonImmuno; Figure 2) or AlexaFluor 488 conjugated Donkey anti-mouse at 1.25 mg/ml (JacksonImmuno; Figure 8). Stained cells were then washed three times with PBS, twice with ddH2O and then sealed in Vectashield mounting medium (Vector Labs).
Tubulin was detected using monoclonal mouse 12G10 anti-Tub1 antibody at 1:1000 concentration (Developmental Studies Hybridoma Bank). Cells were incubated with antibody for one hour, rinsed four times with PBS and then incubated with Cy2-conjugated donkey antimouse antibodies at 1:100 dilution for one hour (JacksonImmuno; Figure 2) or AlexaFluor 488 conjugated Donkey anti-mouse at 1.25 mg/ml (JacksonImmuno; Figure 8). Stained cells were then washed three times with PBS, twice with ddH2O and then sealed in Vectashield mounting medium (Vector Labs).
10
Immunohistochemical analysis of MeCP2 in adult male brains
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