Nissl staining solution methylene blue

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA). BRB, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), HAuCl₄. 3H₂O (≥99.9% trace metals basis), BSA, Triton X-100 and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (USA). RIPA lysis buffer, phenylmethanesulfonyl fluoride, hematoxylin-eosin (HE) staining kit and Nissl staining solution (methylene blue) were obtained from Solarbio (China). The antibodies to anti-CD86, anti-CD206, anti-Cleaved Caspase-3 and anti-TNF-α were purchased from Cell Signaling Technology (USA). Anti-Bax, anti-Bcl-2, anti-IL-1β and anti-IL-6 were purchased from Affinity (USA). Anti-β-Tubulin, anti-GAPDH, HRP Affinipure Goat Anti-Mouse IgG and HRP Affinipure Goat Anti-Rabbit IgG were purchased from Proteintech (USA). The Alexa Fluor^®568 goat anti-mouse/rabbit IgG and Alexa Fluor^®488 goat anti-mouse/rabbit IgG were purchased from Invitrogen (USA). 4,6-dimethyl-2-phenylindole (DAPI) was purchased from Abcam (UK). RAW 264.7 cells and VSC 4.1 cells were obtained from the American type culture collection.

At 3 days or 6 weeks after SCI, a 1.5 cm segment of spinal cord, including the injury epicenter and caudal and rostral segments, was dissected and postfixed in the same fixative for 24 h at 4°C (n = 5 per group). After fixation, the tissue blocks were embedded in paraffin. Transverse sections (10 μm thickness) were taken through the middle of the spinal lesion site and adjacent segments, which were defined as the epicenter, rostral, and caudal, and placed on Superfrost Plus Slides (Thermo Fisher Scientific, Bremen, Germany). The samples were stained with Nissl staining solution (methylene blue, Solarbio, Beijing, China), immediately followed by the administration of one to two drops of permanent mounting medium, and covered with a glass coverslip. All sections were digitally scanned at a high resolution using HistoFAXS 3.0 (Tissue Gnostics, Vienna, Austria). The acquired images were viewed using the associated proprietary viewing software (FAXS viewer, Tissue Gnostics, Vienna, Austria). Photoshop CC (Adobe, San Jose, CA, USA) and ImageJ (National Institutes of Health, Bethesda, MD, USA) software was used to process and analyze images.

At 28 days after the operation, animals were anesthetized and then intracardially perfused with 0.9% normal saline followed by 4% PFA. 1 cm of spinal cord tissue centered on the injury point was removed and then fixed, dehydrated, embedded, and sliced. HE and Nissl staining were performed by Hematoxylin-Eosin Staining Kit (Solarbio, Beijing, China) and Nissl Staining Solution (Methylene Blue) (Solarbio, Beijing, China) according to the manufacturer's instructions. All images were captured under an optical microscope (DMI4000B, Leica, Wetzlar, Germany). For Nissl staining, we counted the number of ventral motor neuron (VMN) in spinal anterior horn of sections as described previously [29 (link)].