Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples using density gradient centrifugation (Leucosep, Interpath Services, Somerton, VIC, Australia) and cryopreserved. PBMCs were immunophenotyped to determine proportions of B cell subsets. Anti-mouse compensation beads (eBioscience, San Diego, CA, USA) were used as single colour controls for compensation analysis, and Fluorogold to exclude dead cells (Invitrogen, Carlsbad, CA, USA). All phenotypic data were acquired on a Fortessa X-20 using BD FACSDiva 8.0.1 software, and all acquired flow data analysed using FlowJo 10.7.1. The following antibodies were used: anti-CD10 (HI10a; PE-CF594), anti-CD20 (2H7; BUV395), anti-CD21 (B-ly4; APC), anti-CD24 (ML5; BV605), anti-CD38 (HIT2; PerCP-Cy5.5), anti-IgG (G18-145; PE-Cy7), and biotinylated anti-IgA/G/M (BD Biosciences, Franklin Lakes, NJ, USA); anti-CD27 (M-T271; FITC), anti-IgA (IS11-8E10; PE), anti-IgD (IA6-2; BV510), and anti-IgM (SA-DA4; EF450) (Miltenyi Biotech, Gladbach, Germany); anti-IgM (MHM-88; AF647) (Biolegend, San Diego, CA, USA); goat anti-human IgA/G/M, and streptavidin-HRP (Southern Biotech, Birmingham, AL, USA); and IgA/G/M standard (Sigma-Aldrich, Saint Louis, MO, USA).
Fluorogold
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorogold is a fluorescent dye used for labeling and tracing cells and neuroanatomical structures. It is a water-soluble, non-toxic compound that emits a bright yellow-gold fluorescence when exposed to ultraviolet or blue light.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti cd27 m t271, by Miltenyi Biotec (1 mentions)
Facsdiva, by BD (1 mentions)
Vibratome vt1000, by Leica (1 mentions)
Cm30505 cryostat, by Leica (1 mentions)
Fv300, by Olympus (1 mentions)
Cd38 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd138 pe, by Thermo Fisher Scientific (1 mentions)
Facsaria fusion sorter, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Cm1950, by Leica (1 mentions)
Fluoro ruby, by Thermo Fisher Scientific (1 mentions)
Anti inkt microbeads, by Miltenyi Biotec (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Macs separator, by Miltenyi Biotec (1 mentions)
Alexa fluor 555 conjugate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Snc80, by Bio-Techne (1 mentions)
Cym51010, by Merck Group (1 mentions)
Damgo, by Merck Group (1 mentions)
16 protocols using fluorogold
1
Immunophenotyping of PBMC Subsets
2
Retinal Ganglion Cell Quantification
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Mice were anesthetized as described above, and a 5% solution of the neurotracer dye Fluoro-Gold (Invitrogen, NY, USA) was applied to their superior colliculi using a piece of soaked Gelfoam. Skull openings were then sealed with a petrolatum-based antibiotic ointment. The overlying skin was sutured and antibiotic ointment was applied externally. Seven days after the application of Fluoro-Gold, the eyes were enucleated, and the retinas were detached at the ora serrata and cut with a trephine around the optic nerve head. Four radial relaxing incisions were made, and the retinas were prepared as flattened whole mounts on silane-coated microscope slides29 (link). Fluoro-Gold-labelled RGCs in flat-mounted retinas were counted in 4-mm2 areas, for a total of 16 fields of cells per retina (each field, 500 × 500 μm2) without position-matching.
3
Fluoro-gold Tracing of Mouse Superior Colliculus
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The detailed experiment is well explained in our previous study [17 (link)]. Briefly, the neurotracer dye, Fluoro-gold (5% solution in saline, Invitrogen, NY, USA) was injected to the superior colliculus of mice using a piece of soaked Gelfoam. The eyes were taken out seven days after the Fluoro-gold application, and the retinas were detached at the ora serrata, followed by cutting with a trephine near the optic nerve head. After four radial incisions, the retinal flat mounts were prepared on silane-coated microscope slides.
4
Retrograde Tracing of BLA-ACC Afferents
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Analysis of afferent neurons from the BLA to the ACC was performed by injecting the retrograde tracer hydroxystilbamidine methanesulfonate (FluoroGold®, Molecular Probes, 0.5 µl) bilaterally in the ACC, using a microsyringe pump controller (UMC4, World precision instruments) and a 5-µl Hamilton syringe (100 nl/min, coordinates for the ACC, AP: + 0.7 mm from bregma, L: + /−0.2 mm, DV: −2mm from the bregma). 7 days after the tracer injection, mice were anesthetized with Euthasol (182 mg/kg) and perfused with 30 ml of 0.1 M phosphate buffer (PB, pH 7.4) followed by 150 ml of 4% paraformaldehyde solution (PFA) in 0.1 M PB. Brains were removed, postfixed overnight in PFA at 4 °C, and then kept at 4 °C in 0.1 M PB saline (PBS, pH 7.4) until cutting. Coronal sections (40 µm) were obtained with a Vibratome (VT 1000 S, Leica, Deerfield, IL) and serially collected in PBS.
5
Bone Marrow Cell Flow Cytometry
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The following antibodies were used for flow cytometric analysis of cells isolated from bone marrow: APC-conjugated anti-B220/CD-45R (eBioscience), fluorescein isothiocynate (FITC)-conjugated anti-mouse IgM, and PE-conjugated anti-mouse IgD (BD Pharmingen). Stained cells were resuspended in buffer containing 2 μmol/L FluoroGold (hydroxystilbamidine, Molecular Probes, Invitrogen) before sorting on a FACSAria II flow cytometer (BD Biocsiences, San Jose, USA).
6
Retrograde Labeling of DRG Neurons
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L4 and L5 DRG neurons were retrograde labeled with a fluorescent dye, Fluoro-Gold (Molecular Probes, Eugene, OR, USA) at 4 weeks postoperatively. After anesthesia, the crushed sciatic nerves were cut, and Fluoro-Gold was put on the distal side of the proximal part of the sciatic nerve and then sutured. After 5 days, the rats were deeply anesthetized and perfused with saline and paraformaldehyde as described previously (Savignat et al., 2008; Li et al., 2012). The ipsilateral L4 and L5 DRGs were removed and postfixed overnight with paraformaldehyde. DRGs were immersed in a 20% sucrose solution for 4 days, embedded in Tissue Tek (Sakura, Tokyo, Japan), and frozen in liquid nitrogen. Serial 35 μm longitudinal sections were cut at −20°C using a cryostat microtome (CM30505 Cryostat, Leica). Sections were then observed under a fluorescence microscope equipped with a rhodamine filter (Olympus FV-300). The number of Fluoro-Gold-positive neurons in each DRG was counted. The positive-labeled neurons at the DRG section of each group were randomly selected, and their area (soma size of neuron) was measured and averaged using computer software (OPTIMAS Ver. 6.5) (Savignat et al. 2008).
7
Purification of Plasma Cells and Non-Tumor Cells
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PCs and non-tumour cells were purified using multicolour flow cytometry as previously described [21 (link)]. Briefly, ~1 × 105 mononuclear cells were prepared for single-stain antibody control (CD138-PE (Beckman Coulter #A54190) and CD38-PE-Cy7 (Biolegend #303515)) and compensation/FMO tubes (1: unstained; 2: hydroxystilbamidine (FluoroGold; Life Technologies) only; 3: CD38-PE-Cy7 + FluroGold; 4: CD138-PE + FluroGold; and 5: CD38-PE-Cy7 + CD138-PE). The sort sample was stained with CD138-PE and CD38-PE-Cy7 antibody at 1 μL/100 μL cells. Cells were stained with FluoroGold immediately prior to sorting. Viable PCs (CD138+CD38+ and FluoroGold negative) and non-tumour cells were sorted on the FACSAria Fusion sorter (BD Biosciences). FACS purity check was carried out on sorted cells, using 100–500 cells from each sample.
8
Trigeminal Innervation Labeling of MCA
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Labeling of the trigeminal innervation of the middle cerebral artery (MCA) followed the procedure of O’Connor and van der Kooy.9 Briefly, male rats were anesthetized with ketamine/xylazine (80 and 10 mg/kg body weight, respectively) and a cranial window prepared. The dura was cut and reflected away to expose the right MCA and a small piece of parafilm was positioned underneath the artery. Gelfoam soaked in a 10% solution of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Life Technologies) dissolved in ethanol was placed on top of the artery after which another piece of parafilm was placed on top of the gelfoam. The skin was sutured and the animal was placed on a heating pad during recovery. During recovery, 5 µl of hydroxystilbamidine (Fluorogold, 10% solution in DMSO; Life Technologies) was slowly administered into the right nasal cavity of the rat to label trigeminal innervation of the nasal epithelium. Animals were allowed to recover for one week prior to inhalation experiments. In addition, some of the animals received an injection of DiI into the hindpaw footpad during recovery to label neurons in the dorsal root ganglia.
9
Immunophenotyping of Hematopoietic Cells in Mice
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Approximately 500 μl of peripheral blood was obtained from mice by cardiac puncture and collected in microfuge tubes containing 50 μl of 0.5 M EDTA. Femora and tibiae were flushed and BM cells were collected. Red blood cells were removed by hypotonic lysis and leukocytes were stained with phycoerythrin (PE)-Cy7–conjugated rat anti-mouse B220 (eBioscience, San Diego, CA), fluorescein isothiocyanate–conjugated rat anti-mouse CD3 (eBioscience), PE-Cy5–conjugated rat anti-mouse CD11b (BioLegend, San Diego, CA), APC-conjugated rat anti-mouse Gr1 (eBioscience), and PE-conjugated rat anti-mouse NK1.1 (BD Biosciences, San Jose, CA). FluoroGold (Life Technologies) was used to exclude dead cells. Cells were sorted on a FACSAria II (BD Biosciences) into B cell (B220+), T cell (CD3+NK1.1−), monocyte (CD11bhiGr1lo), and granulocyte (CD11bhiGr1hi) populations for RNA extraction and real-time PCR. PCs were isolated from flushed long bones and identified using rat anti-mouse CD138 (R&D Systems, Minneapolis, MN) followed by PE-conjugated goat anti-rat IgG.
10
Retrograde Labeling of Spinal Neurons
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Three SD rats were taken from each group for the tracing and labeling experiment, and the method proposed in (12 (link)) was adopted. The regenerated sciatic nerve of rats was re-exposed and cut off. The distal ends of the common peroneal nerve and tibial nerve were immersed in 5% Fluoro-Ruby (FR, Life Technologies) and 4% Fluoro-Gold (FG, Life Technologies) for 2 h, respectively. The wound was washed and sutured layer by layer. Then rats were fed for 7 days so that the retrograde tracer could label the spinal cord neurons. After anesthetizing, the heart of rats was isolated and perfused. L4–L6 lumbar spinal cord segments containing sciatic nerves were cut off and fixed in 4% paraformaldehyde for 12 h. The sample was dehydrated in 10, 20, and 30% sucrose for 12 h, and then sliced to a 40 μm-thick piece by a frozen slicer (Leica CM 1950, Germany). Subsequently, the sample was placed on the glass slide and sealed, and the slice was imaged by the 20 × microscope objective. The excitation wavelength was FG330nm and FR561nm. Finally, the acquired fluorescence image was analyzed using Image J software to detect the neurons labeled by FG only, FR only, and both FG and FR.
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