RGCs were retrogradely labeled by injecting a solution of DiI fluorescent tracer (Molecular Probes) or Fluoro-Gold (FG, Sigma) into the superior colliculus 3 or 7 days before euthanasia, as described previously (Wu et al., 2010 (link)). At euthanasia, the eyes were enucleated, and the retinas were prepared as flat mounts. RGC counts were performed, as previously described (Wu et al., 2010 (link)). Cell counting was performed independently by 3 observers in a double-blinded fashion. The data are expressed as the relative percentage of RGC loss in the experimental eye compared with that in the contralateral control.
Fluoro gold fg
Manufactured by Merck Group
Sourced in United States
Fluoro-Gold (FG) is a fluorescent tracer compound used in neurological research. It is a retrograde neuronal tracer that allows the visualization and identification of neurons that project to a specific region in the brain. FG is resistant to fading and can be detected using fluorescence microscopy techniques.
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Lab products found in correlation
Axio imager 2, by Zeiss (1 mentions)
Ctb alexa 555, by Thermo Fisher Scientific (1 mentions)
Cm1950, by Leica (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Dm6000b, by Leica (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Sucrose, by Fujifilm (1 mentions)
Isoflurane, by Fujifilm (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
9 protocols using fluoro gold fg
1
Retrograde Labeling of Retinal Ganglion Cells
2
Retrograde RGC Labeling and Quantification
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The procedures were performed as previously reported (Wu et al., 2013 (link)). Briefly, Fluoro-Gold (FG, Sigma–Aldrich, St. Louis, MO, USA) was injected into the superior colliculus on each side after the rats in each of the four groups were deeply anesthetized. One week post-injection, the rat eyes were harvested and placed in 4% PFA for 2 h at room temperature, and the whole retina was then carefully dissected and flat-mounted on a slide. The RGCs were counted and averaged per eight microscopic fields of identical size at a distance of 1–2 mm from the optic nerve head where the RGC densities were comparable at 200 × magnification. Each group contained at least three rats for measurement of the mean density. The RGCs were manually counted by two operators in a blinded manner using ImageJ software (NIH, Bethesda, MD, USA). The density of RGCs is expressed as the number of cells per mm2.
3
Evaluating Retinal Ganglion Cell Neuroprotection
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Seven days before sacrifice, the rats were deeply anesthetized. Then, 2 μl of 5% of FluoroGold (FG; Sigma-Aldrich, St. Louis, MO, USA) was injected into the superior colliculus on each side, as previously reported (Wu et al., 2013 (link)). At euthanasia, the eyeballs were enucleated and directly fixed in 4% paraformaldehyde for 2 h at room temperature. The retinas were then carefully dissected and prepared as flatmounts. RGCs were quantified and averaged per eight microscopic fields of identical size using a laser scanning confocal microscope (TCS SP8, Hamburg, Germany) at a final magnification of 200×. The RGCs were manually counted by two operators who were blinded to the study using ImageJ software (NIH, Bethesda, MD, USA). The RGC density is expressed as the number of cells per mm2.
FG labeling indicates that the protective effect is most obvious at 2 weeks after Qcn administration, to further clarify the protective effect of Qcn on RGCs under COHT, we performed TUNEL and survivin staining on retinal cryosections at 2 weeks after COHT.
FG labeling indicates that the protective effect is most obvious at 2 weeks after Qcn administration, to further clarify the protective effect of Qcn on RGCs under COHT, we performed TUNEL and survivin staining on retinal cryosections at 2 weeks after COHT.
4
Facial Nerve Regeneration Tracking
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Fluorescent tracers were injected into the paralyzed/reinnervated facial muscle to retrogradely detect the regenerated motoneurons in the facial and hypoglossal nuclei. Rats were anesthetized as in the initial surgery, and 10 μl of 2.5% Fluoro-Gold (FG; Sigma) was injected into the right orbicularis at multiple points, while 20 μl of 1% cholera toxin subunit B conjugated to Alexa Fluor 555 (CTB-Alexa 555; ThermoFisher) was injected into the right whisker pad. One week later, rats were killed by intraperitoneal overdose injection of pentobarbital (120 mg/kg). Intracardiac perfusion with 300 ml of phosphate-buffered saline (0.1 M, pH 7.2) followed by 200 ml of 4% paraformaldehyde (PFA) was then performed. The brainstem was removed and preserved in 4% PFA solution for 3 h. After gradient dehydration in sucrose solutions, specimens were immersed in optimal cutting temperature (OCT; Sakura, USA) compound and cross-sectioned at a thickness of 25 μm using a freezing microtome (CM1950; Leica, Germany). Fluorescently labeled motoneurons were identified and counted using a Zeiss Axio Imager 2 imaging optic fluorescence microscope (Axio Imager M2, Zeiss, Germany).
5
Retrograde tracing of PVN neural circuits
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After anesthetization, rats were mounted on a stereotaxic apparatus. Retrograde tracing from the PVN followed unilateral pressure injection of Fluoro-Gold (FG, Sigma-Aldrich Chemical, MO, United States; 2% in 0.9% NaCl). Pressure injections were performed using a microinfusion pump with a volume of 200 nL. After a 7-day recovery, rats were perfusion-fixed with 4% paraformaldehyde. The brains were removed for post-fixing, dehydrating, embedding, and sectioning. Photographs of the fluorophores were taken under a Leica DM6000B fluorescence microscope (Leica Microsystems AG, Wetzler, Germany).
6
Optic Nerve Transection and Fluorogold Labeling
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Same way as ONT to expose optic nerve, a complete transection was made to the ON 1.5 mm posterior to the optic disc. A piece of gel foam soaked with 6% Fluoro-Gold (FG) (Sigma, HK) was placed at the proximal optic stump. Care was taken to maintain the blood supply to the retina.
7
Retrograde Tagging of Retinal Ganglion Cells
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Retrograde tagging of RGCs was conducted 7 days before the introduction of I/R in rats, as reported in previous studies (Fu et al. 2021; Pang et al. 2020) . A 5% solution of Fluoro-Gold (FG) (Sigma-Aldrich, St. Louis, MO, USA) was used for RGC labeling. Briefly, 2 mL of FG was introduced into the superior colliculi of animals under anesthesia assisted by a stereotaxic apparatus to ensure zero moments. After 7 days of FG labeling, a retinal I/R insult was induced following the procedures mentioned earlier (Guruvaiah et al. 2018 , Wu et al. 2019 ).
After the study, the eyeballs of each animal were enucleated after euthanasia and preserved for 1 hr at ambient temperature in 4% paraformaldehyde. Retinal tissue samples were meticulously sliced and flat-mounted on glass slides using 10% glycerol in PBS (Sigma-Aldrich Corp. China) after washing with PBS. Images of FG-labeled RGCs were captured using a fluorescence microscope (Olympus Optical, Tokyo, Japan). RGCs were calculated in
After the study, the eyeballs of each animal were enucleated after euthanasia and preserved for 1 hr at ambient temperature in 4% paraformaldehyde. Retinal tissue samples were meticulously sliced and flat-mounted on glass slides using 10% glycerol in PBS (Sigma-Aldrich Corp. China) after washing with PBS. Images of FG-labeled RGCs were captured using a fluorescence microscope (Olympus Optical, Tokyo, Japan). RGCs were calculated in
8
Immunohistochemical Analysis of CGRP
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MIA, fluorogold (FG), and paraformaldehyde (PFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-human CGRP polyclonal antibody was purchased from Peninsula Laboratories LLC (San Carlos, CA, USA). Isoflurane, sucrose, and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Mayer’s Hematoxylin and Eosin were purchased from Muto Pure Chemicals Inc. (Tokyo, Japan).
9
Retrograde Tracer Injection for Spinal Cord Injury
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At 6 weeks after contusion, the remaining rats from each group were anesthetized as described above and the T10 injury site was exposed again. Fluorogold (FG, 1 µL, Sigma, USA) was injected into the spinal cord at four points (0.5 mm depth, 0.3 mm and 0.6 mm bilaterally to the midline, 0.25 µL each), about 5 mm caudal to the contusion site. All injections were performed by the same researcher, to maximize comparability between animals. Muscles were sutured in layers and the skin was closed. Animals were returned to their cages for an additional week.
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