Alexa fluor 555 goat anti mouse igg

Approximately 1,000–2,000 cells were washed twice in cold PBS with 2% FBS and diluted in 400 μL of cold PBS plus 1% FBS. Samples were loaded into wells of Thermo Scientific Cytospin 4 (Thermo Fisher Scientific) and spun at 800 rpm for 5 min. Slides were fixed with either methanol/acetic acid (v/v 3 : 1) for 3 min or precooled acetone for 10 min and dried for 30 min. Slides fixed by methanol/acetic acid were stained with Wright-Giemsa dye for 5 min, followed by soaking in PBS for 10 min and a quick wash in distilled water. Blood cell typing/morphological criteria were confirmed by Morphology Center of Hematology Department, Cancer Center, The First Clinical Hospital of Jilin University.
Slides fixed by acetone were soaked in distilled water for 5 min, followed by rinsing in PBS buffer for 3 times. Slides were then blocked with 10% FBS for 1 h at room temperature and incubated with primary antibodies (optimal working dilutions for all antibodies were listed in Table 2) for 60 min at 37°C in dark. Slides were then washed with PBS buffer for 3 times. Alexa Fluor-555 goat anti-mouse IgG (1 : 200, Cell Signaling Technology) was used as secondary antibody. After washing by PBS, nuclei were counterstained with 5 μg/mL DAPI for 2 min in dark, and cells were visualized with a laser scanning confocal microscope.

Double labeling with FJC and βIII-tubulin (a neuronal cell marker) was performed to detect degenerating neurons. Immunofluorescence staining was performed as described above. The tissue slides were incubated with mouse anti-βIII-tubulin (1:200; R&D system, # MAB1195) at 4 °C overnight, followed by incubation with Alexa Fluor 555 goat anti-mouse IgG (1: 1000, Cell Signaling, #.4409) for 1 h at room temperature. FJC staining was performed with the Biosensis’ ready-to dilute FJC staining kit (Biosensis, Tebarton, South Australia) according to the manufacturer’s protocol. Briefly, all fluorescently labeled slides were immersed in 10% potassium permanganate (v/v) for 5 min and then rinsed in distilled water for 2 min twice. Subsequently, the slides were incubated with 20% FJC and 20% DAPI (v/v) in 0.1% acetic acid solution for 20 min at room temperature. After washing with distilled water, the slides were dried and covered with a coverslip using DPX mounting media (Sigma-Aldrich).

RNAlater and HotStarTaq® Master Mix Kit were purchased from Qiagen (Valencia, CA, USA). TRizol Reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 5 X All-In-One RT MasterMix (with AccuRT Genomic DNA Removal Kit) was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada). RIPA buffer, BCA Protein Assay kit, Chemiluminescent Substrate Detection kit and HRP-linked goat anti mouse IgG were purchased from Boster (Wuhan, China). Protease inhibitor cocktail and phosphatase inhibitors were purchased from Roche (Basel City, Switzerland). Rabbit antibodies against VGluT1, EAAT2, tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and mouse antibody against VGluT3 were all purchased from Abcam (Cambridge, UK). Rabbit antibodies against VGluT2, EAAT1, EAAT3, β-actin and mouse antibody against GFAP, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Diaminobenzidine, isoflurane and mouse anti-TH antibody were ordered from Sigma (St Louis, MO, USA). Polink-2 plus® Polymer HRP Detection System was obtained from ZSGB-BIO (Beijing, China).

RNAlater, the QuantiTect Reverse Transcription Kit and the HotStarTaq^® Master Mix Kit were purchased from Qiagen (Valencia, CA, United States). Trizol reagent was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Rabbit anti-BACE1 and mouse anti-neurofilament (NF) antibodies were all purchased from Abcam (Cambridge, MA, United States). Mouse anti-GFAP, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Waltham, MA, United States). The GTVisionTM III Detection System/Mo & Rb Kit and the DAB Detection Kit were purchased from Gene Tech (Shanghai, China). Isoflurane, a mouse anti-TH antibody and manganese (III) 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis (methochloride) (MnTMPyP) were ordered from Sigma (St. Louis, MO, United States).