Paraffin embedding center

The whole brains were dissected 24 h after MCAO surgery and cut into 5 mm thickness. The brain tissues were fixed in 4% neutral buffered paraformaldehyde solution and washed overnight with tap water. They were dehydrated with graded ethyl alcohol (70% to 100%), cleaned with xylene, and embedded in paraffin using a paraffin embedding center (Leica, Wetzlar, Germany). The paraffin blocks were cut into 4 μm thickness using a rotary microtome (Leica), mounted on slide glasses, and dried in a slide warmer (Thermo Fischer Scientific, Waltham, MA, USA). The slides were deparaffinized in xylene, rehydrated with graded ethyl alcohol (100%-70%), and transferred to tap water. They were stained in Harris’ hematoxylin solution (Sigma-Aldrich) and washed with running tap water. They were differentiated in 1% hydrochloric acid solution with 70% ethyl alcohol, dipped in tap water, neutralized in 1% ammonia solution, and immersed in tap water. The slides were stained with eosin Y solution (Sigma-Aldrich), dehydrated with graded ethyl alcohol (70% to 100%), and rinsed with xylene. They were coverslipped with permount mounting medium (Thermo Fischer Scientific) and the right cerebral cortical region was observed and photographed with an Olympus microscope (Olympus, Tokyo, Japan).

Fixed brain tissues were washed with running tap water for overnight. Washed tissues were dehydrated in gradient ethyl alcohol (70 to 100%) and cleaned with xylene. Tissues were embedded in paraffin with the paraffin embedding center (Leica, Westlar, Germany). Embedded tissues were cut into 4 μm thickness slices and placed over glass slides. Tissue slides were kept on slide warmer for drying, deparaffinized with xylene, and rehydrated with gradient ethyl alcohol (100 to 70%). Tissue slides were stained with Harris’ hematoxylin solution (Sigma) for 3 min and washed with running tap water for 10 min. They were dipped in 1% hydrochloric acid solution and 1% ammonia water, dipped in tap water, and stained with eosin Y solution (Sigma) for 3 min. Tissue slides were washed with tap water, dehydrated with gradient ethyl alcohol (70 to 100%), cleaned with xylene, and coverslipped with permount solution (Thermo Fisher Scientific, Waltham, MA, USA). They were observed under optical microscope (Olympus, Tokyo, Japan) and images were taken from cerebral cortex area.

Brain tissues were fixed in 4% neutral buffered paraformaldehyde, washed with tap water for overnight, dehydrated with graded series of ethanol from 70 to 100%, and cleaned with xylene. They were infilterated with paraplast (Leica, Wetzlar, Germany) and embedded using paraffin embedding center (Leica). Paraffin blocks were cut into 4 µm thicknesses using rotary microtome (Leica) and paraffin ribbons were mounted on slide glass. Sections were dried on slide warmer (Thermo Fisher Scientific, Waltham, MA, USA). They were dipped in xylene to remove paraffin, hydrated with graded series of ethanol from 100 to 70%, and washed with tap water. Sections were stained with Harris’ hematoxylin solution (Sigma-Aldrich) for 5 min and washed with tap water. They were subsequently dipped in 1% hydrochloric acid with ethanol, washed with water, and dipped in 1% ammonia water. Sections were washed with water, stained with eosin Y solution (Sigma-Aldrich) for 2 min, and washed with water. They were dehydrated with graded series of ethanol from 70 to 100%, cleaned with xylene, and coverslipped with mounting solution (Thermo Fisher Scientific). The stained tissues were observed and photographed under an Olympus microscope (Olympus, Tokyo, Japan).