Axiom1

To investigate morphological alterations, CV staining was performed according to a previously published procedure (24 (link)). Briefly, the sections were stained with 1% CV acetate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and immersed in serial ethanol baths. CV-stained structures were observed under an AxioM1 light microscope (Zeiss AG, Oberkochen, Germany) equipped with a camera (Axiocam; Zeiss AG) and photomicrographs were captured. The CV-stained structures were examined in a 250×250 µm area that included the stratum pyramidale at the center of the hippocampal CA1 region, or in the whole dentate gyrus, using the image analysis system Optimas version 6.5 (CyberMetrics, Scottsdale, AZ, USA).

NeuN- and F-J B-positive cells were counted according to our published procedure (Bae et al., 2015). Fifteen brain tissue sections were chosen in each animal with 120 μm interval. NeuN- and F-J B-positive cells in 200 × 200 μm2 at the center of the CA1 stratum pyramidale were counted using an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Axiocam, Carl Zeiss) interlinked with a PC monitor. Cell counts were analyzed as a percent, with the sham group and ischemia group (5 days) designated as 100%. To quantitatively analyze CB immunoreactivity, in brief, according to our method (Lee et al., 2016), images were calibrated into an array of 512 × 512 pixels corresponding to a tissue area of 140 × 140 μm2 (40× original magnification). The mean CB immunoreactivity was determined in hippocampal CA1 pyramidal neurons by a 0–255 gray scale system in ImageJ (National Institutes of Health, MD, USA). The background density was subtracted, and the relative immunoreactivity (RI) of image file was calibrated as % using Adobe Photoshop version 8.0, and the RI was analyzed using NIH Image 1.59 software (National Institutes of Health). RI was calibrated as %, with sham group designated as 100%.

The brain tissues were stained with Fluoro-Jade B (F-J B) which has a high affinity for dead neuron [36] (link), [37] (link). The sections attached on the gelatin-coated slide glass were incubated in a 1% sodium hydroxide solution, 80% ethanol for 5min, and hydrated in 70% ethanol-distilled water. The tissue slices were treated with a 0.06% potassium permanganate solution and shaking on a horizontal shaker for 10 min. The tissue samples were washed in distilled water and incubated in a 0.0001% F-J B (Histochem, Jefferson, AR, USA) working solution for 30 min. The stained-sections were rinsed in the distilled water and completely dried in the dry oven. The dried sections were sealed with DPX (Sigma, USA), and the image of tissue was observed by an epifluorescence microscope (AxioM1, Carl Zeiss, Göttingen, Germany) with 450–490 nm light.