Following deep anesthesia, the rat heads were fixed in a stereotactic apparatus (Stoelting Kiel, Germany) and the skin covering the skull of the rats was incised. Fluoro-Gold (FG;Biotium, Hayward, CA, USA) was injected (2 μl of 4% FG in distilled H2O) into the superior colliculus (SC) on each side using a microsyringe, and was retained for 10 min. The animals were maintained for 1 week post-labeling and subsequently the eyes were enucleated and fixed with 4% PFA for 1 h. The retinas were examined with an Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan) with UV excitation (excitation filter, 350–400 nm; barrier filter, 515 nm) and a digital imaging system. The RGCs were examined by division into 4 quadrants (superior, inferior, nasal,and temporal), which were further divided into central (0.8–1.2 mm from the optic disc), middle (1.8–2.2 mm from the optic disc), and peripheral regions (0.8–1.2 mm from the retinal border). A total of 2 standard square areas (200 × 200 μm2) were measured in each region. The density of RGCs in each group of rats was expressed as the number of labeled RGCs/mm2 compared with the counted retinal area [14 (link)].
Fluorogold fg
Manufactured by Biotium
Sourced in United States
Fluorogold (FG) is a fluorescent retrograde tracer that is used in neuroscience research to label and visualize neurons. It is a water-soluble compound that emits a yellow-gold fluorescence upon exposure to ultraviolet or blue light.
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9 protocols using fluorogold fg
1
Retrograde Labeling of Retinal Ganglion Cells
2
Retrograde Labeling of Spinal Cord Projection Neurons
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In the 7th postoperative week, the rats (n = 5 per group) were randomly selected for retrograde labeling of host axonal tracts. The cell bodies of neurons projecting to the spinal cord caudal to the transplanted area were labeled with Fluorogold (FG; Biotium, San Francisco, USA). Briefly, a dorsal laminectomy was performed at T12, and 0.5 μl of FG was injected into the spinal cord using a Hamilton syringe. At 1 week after injection, the animals were sacrificed, and the T8 segment of the spinal cord was removed, cryopreserved in graded sucrose solutions, and sliced into 10 μm frozen sections. The sections were observed via a confocal laser scanning microscope (880, Carl Zeiss AG, Germany) to detect and count FG-labeled neurons in the ventricles. The number of FG-labeled axons was assessed by two independent examiners who were blinded to the rats’ treatment status. The rats injected with FG were not used for functional evaluation.
3
Retrograde Tracing of Spinal Axons
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Seven weeks after surgery, the rats were randomly selected to assess the retrograde labeling of axonal tracts by Fluorogold (FG; Biotium, San Francisco, CA, USA). Briefly, spinal cords were exposed at T12, and 0.5 μL of FG was injected into the exposed cord segment using a Hamilton syringe. One week after injection, the rats were sacrificed, and the T8 cord segment was removed (Chen et al., 2020), cryopreserved and dehydrated in graded sucrose solution, and sliced into 10-μm frozen sections in the transverse plane. The sections were viewed via a fluorescence microscope (DM6B, Leica) and FG-labeled neurons in the ventricolumna were counted. These rats were not used for functional evaluation.
4
Fluoro-gold Injection and Dural Infusions
5
Retrograde Tracing After Spinal Cord Injury
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Animals (N = 5 per group) were used for retrograde tracing at 7 weeks after SCI. Dorsal laminectomy was performed at T12, and 0.5 μl of Fluorogold (FG; Biotium, Fremont, CA; #80014) was injected into the spinal cord using a Hamilton syringe. One week after injection, the animals were perfused and the T7 segment of the spinal cord was removed, cryopreserved, embedded in OCT compound, and sliced into 10 µm frozen sections. A fluorescence microscope (Olympus, Tokyo, Japan) was used to detect FG-labeled neurons.
6
Fluoro-gold Injection and Dural Infusions
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Similar to virus injections, Fluoro-gold (FG, 55.2 nL 4% in H20, Biotium, Fremont, CA) was injected into the right VTA (AP −5.8, DV −8.5, ML 0.5 mm from bregma) of animals weighing 275-300 g. In the same surgery, a dural cannula was placed for IM infusions as described above. One week after FG injection, animals underwent microinjection of PBS or IMs into dural cannulas. Two hours after injection, animals were deeply anesthetized and perfused.
7
Rat Retinal Explant Preparation and RGC Labeling
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The study protocol has been approved by the Local Committee for an Animal Research and follows the ARVO statement for the use of Animals in Ophthalmic and Vision Research. In all experiments, we used approximately eight-week-old male Wistar rats weighing approximately 180 g (Center of Experimental Medicine, Medical University of Silesia, Katowice, Poland). For the retinal explant preparation, we used 20 animals. Twelve of them received a 3 μl injection of 3% hydroxystilbamidine (FluoroGold, FG, Biotium, Fremont, CA, US) in 10% DMSO-saline into both the superior colliculi of the midbrain seven days before animals were sacrificed (FLOREC group). The specific contents of the injection allowed us to retrogradely label the retinal ganglion cells (RGC) [32 (link)]. The FG injection was performed under the general anesthesia with an intraperitoneal injection of ketamine (50 mg/kg; VetaKetam, Vetagro, Poland) and xylazine (5 mg/kg; Xylapan, Vetoquinol Biowet, Poland). The sites of injection were localized on the rat skull using stereotactic equipment. To ensure the correct localization of injection, the online atlas of the rat brain was used (Figure 1 ). Other eight animals were utilized without prior FG injection (OREC group).
8
Trigeminal Ganglia Neuron Labeling
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To label trigeminal ganglia corneal-projecting neurons, 0.5 μL of 4% Fluorogold (FG, Biotium) or 5 mg/mL wheat germ agglutinin-Alexa Fluor ® 555 (WGA, Life Tech) in PBS was injected into the corneal stroma with Nanofil syringe. [28] Mice were euthanized for trigeminal ganglia immunohistochemistry staining or cell sorting three days after injection.
Fluorogold-labeled trigeminal ganglia were fixed in 10% formaldehyde for 4 hours, embedded in Tissue-Tek OCT compound, and frozen in liquid nitrogen. Next, 6 micrometer-thick sections were cut and mounted to polylysine-coated glass slides. Slides were blocked with 10 mM sodium phosphate buffer containing 2% BSA for 1 hour at room temperature. Sections were then incubated with mouse anti-TRPV1, -TRPV4 (Alomone Labs), or -SP (EMD Millipore, MA). This was followed by a secondary antibody, FITC -conjugated goat antihamster and cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), and slides were mounted with Vectashield mounting medium and examined under confocal microscopy. Controls were similarly treated, but the primary antibodies were replaced with nonspecific IgG.
Fluorogold-labeled trigeminal ganglia were fixed in 10% formaldehyde for 4 hours, embedded in Tissue-Tek OCT compound, and frozen in liquid nitrogen. Next, 6 micrometer-thick sections were cut and mounted to polylysine-coated glass slides. Slides were blocked with 10 mM sodium phosphate buffer containing 2% BSA for 1 hour at room temperature. Sections were then incubated with mouse anti-TRPV1, -TRPV4 (Alomone Labs), or -SP (EMD Millipore, MA). This was followed by a secondary antibody, FITC -conjugated goat antihamster and cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), and slides were mounted with Vectashield mounting medium and examined under confocal microscopy. Controls were similarly treated, but the primary antibodies were replaced with nonspecific IgG.
9
Fluorogold Labeling of Corticospinal Tract
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At P20, the spinal cord was exposed at the C3-C4 level by laminectomy under anesthesia (n = 5). One microliter of 2% (w/v) Fluoro-Gold (FG; Biotium, Hayward, CA, USA) was injected into the center of the dorsal corticospinal tract (1-mm deep from the dura) with a glass micropipette (tip diameter: 60-80 μm). Rats were anes thetized and perfused with 4% (w/v) PFA 6 days later. Brain sections were cut for histological analysis. FG-labeled cells were detected using an anti-FG antibody. The cell numbers were counted in every eighth brain section in the hindlimb motor-regulating cortex [three sections for each brain, 700 × 700 μm 2 in motor (M1 and M2) and sensory areas].
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