Triton x 100 t8787

DAPI (4′, 6-diamidino-2-phenylindole) (D-1306), the surfactants Tween-20 (P7949), NP40 (NP40S) and Triton X-100 (T8787), potassium cyanate, deoxyribonuclease I (D5025) were from Sigma. Vectashield™ mounting media (H-1000) was from Vector Labs. Non-immune donkey (017-000-121 or horse serum (008-000-121) were obtained from Jackson Immunoresearch Labs. Bovine serum albumin (BP1600100) as well as all general chemicals used for these studies were from Thermo Fisher, San Diego, CA, USA.

Porcine type I collagen was kindly donated from Nippon Ham (Osaka, Japan). Gellan gum (GG) (Mw = 500 kDa) was obtained from Sansho (Osaka, Japan). Fibrinogen (from bovine plasma, F8630), thrombin (from bovine plasma, T4648), bovine serum albumin (BSA, A3294), phosphate-buffered saline powder (PBS, D5652), collagenase from Clostridium histolyticum (type I, C0130), and Triton X-100 (T8787) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (35010CV) was purchased from Corning (Corning, NY, USA). Penicillin, streptomycin, BODIPY™ 500/510 C1, C12 (4,4-Difluoro-5-Methyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid (D3823)), goat anti-mouse secondary antibody Alexa Fluor® 647, and Hoechst 33324 (H3570) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The 4% paraformaldehyde (PFA, 16310245) and mouse anti-human CD31 antibody (M0823) came from Wako Pure Chemical Industries (Tokyo, Japan). The human umbilical vein endothelial cell (HUVEC, C25271) and endothelial growth medium (EGM-2MV, CC-3202) were purchased from Lonza (Basel, Switzerland). The Nile Red compound was purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan). Dulbecco's Modified Eagle's Medium (DMEM) came from Nacalai Tesque Inc. (Kyoto, Japan). Cell-Based Propidium Iodide Solution (10011234) came from Cayman Chemical (Ann Arbor, MI, USA).

ROS detection kit (CA1410; Solarbio, Beijing, China) was employed for examining ROS in primary astrocytes. DCFH-DA was diluted with serum-free culture medium according to 1:1000 to make the final concentration 10 μmol/L. After 48 h of drug treatment, astrocytes were incubated with 300 μl DCFH-DA at 37°C for 30 min. Then, astrocytes were washed with PBS to eliminate DCFH-DA which did not enter the astrocytes. In the culture plate, the slides with the climbed cells were immersed in PBS. Primary astrocytes were permeabilized through 0.5% Triton X-100 (T8787; Sigma, USA) at room temperature lasting 20 min as well as the slides were immersed in PBS. After absorbing the PBS with absorbent paper, normal goat serum (C0265; Beyotime, Shanghai, China) was added dropwise on the glass slide, and blocked for 30 min at room temperature. DAPI was added dropwise and incubated for 5 min in the dark to stain the nucleus, and the excess DAPI (D9542; Sigma, USA) was washed away with PBST. Following absorbing the liquid on the slide, the slides were mounted with a mounting solution containing anti-fluorescence quencher, and then observed and collected the images under a BX53 fluorescence microscope (Olympus, Japan).