Samples were initially fixed with 4% paraformaldehyde for 10 mins at room temperature. Then, samples were processed with 0.1% Triton X for 5 mins, followed by 5% BSA blocking for 1 hr at room temperature. The slides were then transferred to the TUNEL (Biotium, 30074) working solution for 1 hr at 37 °C and then rinsed. To combine with Fluoro-Jade, the slides were then transferred to the Fluoro-Jade (Sigma, AG325) working solution for 10 mins and then rinsed, air dehydrated, xylene cleared. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and coverslips were placed. Immunostaining images were obtained with a fluorescence microscope (Nikon ECLIPSE Ti-S) or Nikon A1SiR Confocal Microscope.
Fluoro jade
Manufactured by Merck Group
Fluoro-Jade is a fluorescent stain used for the detection of neurodegeneration in histological samples. It is a sensitive and specific marker for the identification of degenerating neurons.
Automatically generated - may contain errors
Lab products found in correlation
Fluoro jade c, by Merck Group (2 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (2 mentions)
Dpx mounting medium, by Merck Group (2 mentions)
Eclipse ti s, by Nikon (2 mentions)
A1sir confocal microscope, by Nikon (1 mentions)
Axioimager m2 microscope, by Hamamatsu Photonics (1 mentions)
Gb21303, by Wuhan Servicebio Technology (1 mentions)
Gb11138, by Wuhan Servicebio Technology (1 mentions)
5 protocols using fluoro jade
1
TUNEL and Fluoro-Jade Staining Protocol
2
Fluoro-Jade C Protocol for Neurodegeneration
3
Fluoro-Jade C Protocol for Neurodegeneration
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The tissue cryostat sections were mounted on Superfrost Plus microscope slides (FisherScientific, 22–230-892) and dried at 50 °C for 30 min. The slides were immersed in the following solutions at room temperature in order: 0.2% NaOH in 80% ethanol for 5 min, 70% ethanol for 2 min, distilled water for 2 min, 0.06% potassium permanganate for 10 min, distilled water for 2 min, 0.0001% Fluoro-Jade C (Sigma-Aldrich, AG325) in 0.1% acetic acid for 10 min and 3 times distilled water for 1 min. The slides were dried at 50 °C for 10 min, immersed in xylene for 1 min and coverslipped with DPX Mounting medium (Millipore Sigma, 06522).
For double labeling, regular Immunofluorescence protocol as described above was performed first, followed by the Fluoro-Jade staining protocol.
For double labeling, regular Immunofluorescence protocol as described above was performed first, followed by the Fluoro-Jade staining protocol.
4
Fluoro-Jade and H&E Staining of DRG and Spinal Cord
5
Quantification of Neuronal Loss in I/R
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Neuronal loss was quantified as previously described.101 (link) Briefly, 3 and 6 h after I/R (R3h, R6h), animals were deeply anesthetized and transcardially perfused with 0.9% NaCl saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Brain sections (4 µm) were processed for NeuN staining with mouse anti-NeuN (1:1000; Servicebio, GB11138) and reacted with conjugate-absorbed goat anti-rabbit Cy-3 (1:300, Servicebio, GB21303) to determine surviving neurons. The sections were dried overnight for Fluoro-Jade (Sigma-Aldrich), which selectively stains degenerating neurons. The slides were immersed for 3 min in 100% ethanol, for 1 min in 70% ethanol, for 1 min in distilled water, and then transferred to a solution containing 0.01% Fluoro-Jade and 0.1% acetic acid (1:10) for 30 min on a shaker. After three 10-min washes, the slides were finally coverslipped. Labeled sections were imaged with a confocal laser-scanning microscope (Nikon ECLIPSE Ti-S). The number of cells in each section was divided by the area sampled, which was determined using imaging probes.
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