Nissl staining solution

After the MWM test, the mice (n = 4) were sacrificed by cervical dislocation and the brains were removed and immersed in 4% paraformaldehyde to fix the brains. The brain tissues were embedded in paraffin and sliced into 5 μm sections using a microtome (Leica, Nussloch, Germany). The sections were stained with hematoxylin and eosin (H&E) to examine the pathological changes in the cortex by intelligent tissue slice imaging analysis system platform (Pannoremic MIDI, 3DHISTECH, Hungary). Three random fields (400×) in the same cortex region from 4 mice in each group were used to evaluate the pathological damage of neurons by a pathologist double-blindly. Besides, Nissl staining is often used to identify neuronal damage [29 (link)]. We further performed the Nissl staining to study neuronal pathological alterations. The sections (n = 4) were treated with Nissl staining solution (Beyotime Biotechnology, China) for 10 min and were differentiated with 95% alcohol. Then the sections were observed and photographed by intelligent tissue slice imaging analysis system platform (Pannoremic MIDI, 3DHISTECH, Hungary). The density of the Nissl bodies was analyzed double-blindly from three random fields (400×) in the same cortex and hippocampal CA1 regions from 4 mice in each group by using the Image-Pro Plus 6.0 automatic analysis system to assess the change of Nissl bodies.

Hippocampal apoptosis was detected by TUNEL fluorescent staining using the One- Step TUNEL Apoptosis Assay Kit according to the manufacturer’s instructions (Beyotime, Shanghai, China). DAPI staining was used for nuclei staining. The staining was observed using fluorescence microscopy in a 200× visual field (Olympus Corporation, Tokyo, Japan).
For histological analysis, hippocampal neurons were observed by Nissl staining according to the specification of the Nissl Staining Solution (Beyotime, Shanghai, China). The sections were observed using a microscope in 100×, 200×, and 400× visual fields (Olympus Corporation, Tokyo, Japan).

Nissl staining was performed to evaluate neurons as described previously (Zhang et al., 2012 (link); Chen et al., 2013 (link)). After washing with PBS, the sections were incubated with Nissl Staining Solution (Beyotime Institute of Biotechnology, Nanjing, China) for 20 min at room temperature. Sections were then dehydrated with ethanol and covered with Permount.

Nissl staining was used to evaluate neuronal damage in the cortex. Rats were sacrificed via excessive i.p. anesthesia. We performed cardiac perfusion with 0.01 M PBS, followed by 250 mL of 4% paraformaldehyde (4°C) as a tissue fixative, and finally removed the contralateral motor cortex. Routine paraffin embedding was performed and 5 μm sections were subsequently generated. Paraffinized sections were soaked in xylene ×3 for 10 min, then dehydrated via an ethanol gradient (95%, 90%, 80%, 70%, and 50% for 5 min each), immersed in Nissl staining solution (Beyotime, China) for 30 min, rinsed quickly with water, ethanol gradient dehydrated, and soaked in xylene ×3 for 2 min. Finally, sections were coverslipped and three randomly selected visual fields were photographed under a microscope. Nissl positive cells were quantified using ImageJ software.

The coronal part of the rat brain was loaded onto slides, air‐dried, and then soaked in xylene for 10 min. After gradient alcohol dehydration, the sections were washed with distilled water and stained in 0.5% Nissl staining solution (Beyotime, Shanghai, China) for 10 min at room temperature. The sections were then immersed in 95% ethanol (pH 4.1) for 5 s, followed by fresh 95% ethanol dehydration for 2 min, xylene clearance for 5 min, and neutral gum (Sigma–Aldrich) sealing. Nissl‐positive neurons showed mottled blue‐purple staining with clear nuclei and blue cytoplasm when viewed under a microscope (Olympus).

After dewaxing and rehydration, paraffin slices were incubated with Nissl staining solution (Beyotime, C0117) at 37 °C for 3 min, and then washed with distilled water and 95% ethanol according to the instructions. After dehydration, transparency and sealing, images were obtained under a microscope (Nikon), and the number of neurons was counted by the ImageJ software (Media Cybernetics).

Pramipexole (HY-17355) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Nissl Staining Solution and ELISA Assay Kit were provided by Beyotime (Shanghai, China). The primary antibodies used in this study were anti-GADPH (ab9485; Abcam, Cambridge, UK), anti-DRD2 (DF10211; Affinity, Shanghai, China), anti-Bax (ab32503; Abcam, Cambridge, UK), anti-NeuN (ab196495; Abcam, Cambridge, UK), anti-Caspase-3 (BS1518; Bioworld, Nanjing, China) and anti-Bcl-2 (ab196495; Abcam, Cambridge, UK). Goat anti-rat IgG (HRP) (SA00001-2; Proteintech, Wuhan Shi, China) was used as the secondary antibody for western blotting.