Immunohistochemistry was conducted to assess neuronal damage in the hippocampus. To evaluate the potential of exosomes in attenuating neuronal apoptosis and neurodegeneration, TUNEL staining (ApopTag Fluorescein In Situ Apoptosis Detection Kit, S7110, Merck, Darmstadt, Germany) and Fluoro-Jade C staining (Biosensis, South Australia, Australia) were performed on day 3 following the manufacturer’s protocol [19 (link),20 (link),21 (link)]. Neuronal integrity was assessed using Nissl staining (0.1% Cresyl violet solution; Muto pure chemical, Tokyo, Japan) during the acute phase (day 3) and chronic phase (day 28). To evaluate the effect of exosomes on activated microglia/macrophages, Iba1 staining was performed (1:1500, 019-19741, Wako, Japan). Neuronal inflammation was assessed using IL-1β (1:500, ab283818; Abcam, Cambridge, UK), IL-6 (1:200, bs-0379R; Bioss Inc., Woburn, MA, USA), and TNF-α (1:1000, ab307164; Abcam, Cambridge, UK) staining for day 7 section. Five non-overlapping ROIs were designated in the hippocampal area. The number of positive signals, area, or luminescence was measured using an automated cell/area counter (BZ-X Analyzer, Keyence Co., Osaka, Japan).
Fluoro jade c
Manufactured by Biosensis
Sourced in Australia
Fluoro-Jade C is a fluorescent stain that can be used to label degenerating neurons in brain tissue sections. It is a sensitive and specific marker for neurodegeneration.
Automatically generated - may contain errors
Lab products found in correlation
Ab307164, by Abcam (1 mentions)
Apoptag fluorescein in situ apoptosis detection kit, by Merck Group (1 mentions)
Bs 0379r, by Bioss Antibodies (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Ab283818, by Abcam (1 mentions)
Kainic acid, by Cayman Chemical (1 mentions)
Indomethacin, by Nacalai Tesque (1 mentions)
Liraglutide, by Novo Nordisk (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Entellan, by Electron Microscopy Sciences (1 mentions)
Potassium permanganate, by Merck Group (1 mentions)
Bz x700, by Keyence (1 mentions)
Mab377, by Merck Group (1 mentions)
6 protocols using fluoro jade c
1
Exosome Effects on Hippocampal Neurodegeneration
2
Kainic Acid-induced Neurodegeneration Model
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Kainic acid (KA, 78050, Cayman Chemicals, Ann Arbor, MI), indomethacin (IND, 19233-51, Nacalai tesque, Tokyo, Japan), liraglutide (2499410G1021, Novo Nordisk, Bagsværd, Danmark), and sitagliptin phosphate monohydrate (SPM, A4036, ApexBio, Boston, MA, USA) were used for animal treatments. Fluoro Jade C (FJC, TR-100-FJ, Biosensis, CA) was used for staining of degenerating neurons. Pre-stained Protein Marker (02525-35, Nacalai tesque, Tokyo, Japan) was used for Western blots. MK-0524 (DP1 antagonist, 1480, Axon MEDCHEM, Groningen, Netherlands), OC000459 (DP2 antagonist, 1913, Axon MEDCHEM, Groningen, Netherlands) were used as DP selective inhibitors.
3
Quantifying Astrocyte Activation and Neurodegeneration
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Double staining 3-D semi-quantitative microscopy was used to determine cerebral astrocyte activation and neurodegeneration by using GFAP and Fluoro-Jade C as described previously (Bian et al., 2007 (link); Takechi et al., 2013b (link),c (link)). Briefly, 20 μm cryosections were incubated with rabbit anti-mouse GFAP (1:200, Abcam) for 20 h at 4°C. The sections were then incubated with goat anti-rabbit IgG conjugated with Alexa488 for 2 h at 20°C. After the sections were dried for 10 min at 60°C, Fluoro-Jade C (Biosensis, SA, Australia) staining was done. The sections were incubated with sodium hydroxide (Solution A) for 5 min. Subsequently the sections were incubated with potassium permanganate (Solution B) for 10 min. Finally, the sections were incubated with Fluoro-Jade C (Solution C) and DAPI (Solution D) for 10 min in dark. Confocal microscopy imaging and the measurement of voxel fluorescent intensity of GFAP and Fluoro-Jade C were done as described above.
4
Fluorescent Staining of Brain Sections
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Brain sections were mounted and dried on a slide warmer. Slides were incubated in a 1:10 solution of sodium hydroxide in 80% ethanol for 5 min. Slides were washed with 70% ethanol for 2 min followed by a wash with distilled water for 2 min. Slides were incubated for 10 min in a 1:10 solution of potassium permanganate (Sigma-Aldrich) in distilled water followed by a wash with distilled water for 2 min. Slides were incubated in Fluoro-Jade C (Biosensis) and 4’,6-diamidino-2-phenylindole (DAPI; double stranding DNA staining; Thermo Fisher Scientific) for 10 min in the dark. Slides were then rinsed three times in distilled water for 1 min per rinse, cleared in xylene, and coverslipped with Entellan (Electron Microscopy Sciences). Sections were imaged using a digital Keyence BZ-X700 light and fluorescent microscope.
5
Neurodegeneration Analysis in Slc19a3 Mice
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The three Slc19a3 KO and KI mice in each group were perfused with PBS under anesthesia with pentobarbital intraperitoneal injection (50 μg/g). Brains were fixed in 4% ice-cold paraformaldehyde/PBS perfusion and fixed at 4°C for 12 h. Next, the olfactory bulb and cerebellum were cut off and embedded in paraffin blocks. Serial 5-μm-thick coronal sections corresponding to images 67–69 in Allen Mouse Brain Atlas (http://www.brain-map.org/ [29 ]), and containing the submedial nucleus of the thalamus (SMT) region were cut and prepared for immunohistochemical staining using anti-NeuN (Chemicon #MAB377; Lot.2592741; 1:100) and anti-glial fibrillary acidic protein (GFAP) (DAKO #Z0334; Lot.096; 1:1000). Neurodegeneration was detected by Fluoro-Jade C–positive cells as described previously [30 (link), 31 (link)]. Fluoro-Jade C staining was performed according to the manufacturer’s instructions (Biosensis). To standardize the anatomical structures found in all sections, grids were set on the SMT of the thalamus (0.13-mm2 circle), ventral anterior-lateral complex of the thalamus (VAL, 0.2-mm2 rectangle), and motor field of the cerebral cortex (0.2-mm2 rectangle) according to a brain map [29 ]. NeuN-immunoreactive cell nuclei in the grid were counted using Image J software.
6
Histopathological Assessment of Hydrocephalus
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After diagnosis, a total of 60 brains (37 normal brains, and 14 mild and nine brains with mild and moderate hydrocephalus, respectively) were embedded in paraffin using standard procedures and sliced coronally into 5-μm thick sections. The sections containing the hippocampus and arcuate nucleus were deparaffinized and stained using hematoxylin and eosin to perform a preliminary evaluation of the histopathology of hydrocephalus. Sections from six hydrocephalus-affected brains that exhibited the most representative pathological findings, in addition to six normal brains, were further analyzed in detail. Based on the preliminary results, the primary somatosensory cortex was evaluated to confirm the effects of hydrocephalus on the neocortex. Degenerating neurons were detected using Fluoro-Jade C staining according to the manufacturer's instructions (Biosensis, Thebarton, Australia). Briefly, the deparaffinized sections were incubated in a potassium permanganate solution for 10 min, and a Fluoro-Jade C solution containing DAPI for 10 min under dark conditions. The slides were dried at 45°C and directly coverslipped with non-aqueous mounting media.
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