Fluoro jade b solution

Fluoro-Jade B (FJB) staining was used to investigate the degeneration/death of neurons. The brain sections prepared previously were incubated in 0.06% potassium permanganate for 10 min, followed by 20 min in 0.004% Fluoro-Jade B solution (Millipore, Merck, Germany) at 37°C. After washing and drying, the slides were mounted with coverslips and viewed under the Olympus microscope. The number of FJB-positive cells in the hippocampal CA1 area was manually counted.

FJB staining was used to detect neuron degeneration in adult HI mice. After drying on a slide warmer for 30 min at 45 °C, the slices were rinsed in 80% ethanol containing 1% sodium hydroxide for 2 min, 70% ethanol for 2 min and washed twice in water. The tissue was incubated in 0.06% KMNO₄ for 10 min at RT and washed 3 times in water. Then the slices were incubated in 0.008% Fluoro-Jade B solution (Merck Millipore, USA) containing 0.1% acetic acid for 20 min at RT and kept from lights in the following procedures. After being washed 3 times in water, the slices were dried on the slide warmer again at 45 °C and rinsed in xylene for 5 min at RT. The slides were covered with DPX mounting solution (Sigma Aldrich, USA) and observed with the fluorescent microscope under 488 nm exciting lights.

Fluoro-Jade B (FJB) staining was performed as described previously16 (link)46 (link). In brief, cerebral tissue sections were kept in 0.06% potassium permanganate for 10 minutes and rinsed in distilled water. The sections were stained in 0.0004% Fluoro-Jade B solution (Merck Millipore) in 0.1% acetic acid, then rinsed in distilled water. After being dried, sections were cleared into xylene and coverslipped.

Left brain hemispheres frozen in OCT were coronally sectioned at 30 μm using a cryostat (Microtome HM 505N). Sections were stored in serial order in a 96-well plate in 1 × PBS with sodium azide at 4°C. Fluorojade-B (FJ-B) staining was used to scan and identify neuronal degeneration at 72 h post-CA. To conform to the stereological standards of systematic random sampling, sections from figure 10 (bregma, 3.24 mm) to figure 155 (bregma, −14.64 mm) in the rat brain atlas (The Rat Brain in Stereotaxic Coordinates, 6th edition) were chosen at a 330 μm interval (one of every twelve sections) covering a total 17,880 μm scanned area of potential neurodegeneration. Selected sections were stained for 20 min in 0.0004% Fluorojade-B solution (EMD Millipore, Billerica, MA, United States) after 10 min incubation with 0.06% potassium permanganate. Sections were screened under the microscope and those with Fluorojade-B positive neurons were marked for cell counting. To further avoid biased sampling, two additional sections were chosen at −90 μm anterior and +90 μm posterior of the original marked section.