Alexa fluor 568

Immunofluorescence staining was performed to detect the expression of IGF2BP2 and IGF2BP3. In brief, the tissue samples were deparaffinization, blocked at room temperature for 60 min with a blocking solution (5% BSA/0.1% Triton X-100 PBS), and stained with the primary anti-IGF2BP2 (1:200; 11601-1-AP; Proteintech) and anti-IGF2BP3 (1:200; 14642-1-AP; Proteintech) antibodies. The samples were then washed with Tris-buffered saline for 30 min, and incubated with the secondary goat anti-rabbit IgG H&L (Alexa Fluor^® 568) (1:200, ab175471; Abcam) and goat anti-mouse IgG H&L (Alexa Fluor 488) (1:200, ab150113; Abcam) antibodies for 30 min at 37 °C in the dark. After washing in PBS, DAPI staining was performed for 5 min, to counterstain the nuclei (28 (link)). Finally, the samples were sealed in 50% glycerin and imaged on a fluorescence microscope (DP72, OLYMPUS, Japan).

After fixation in acetone for 10 min, the sections were treated with normal donkey serum for 50 min. Subsequently, the sections were incubated with primary antibody (p-GSK3β, PTEN, and cleaved caspace-3, respectively) at 4°C overnight. The next day, the sections were then incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 (1 : 200; Abcam), followed by nuclei counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, 2 mg/ml). The slides were then examined using a fluorescence microscope (Leica TCS SP5).

BEAS-2B cells were seeded on confocal dishes and exposed to the vehicle (water), B[a]P (5 μM) alone, B[a]P (5 μM) plus H.cordata (12.5, 25, or 50 mg/mL), or B[a]P (5 μM) plus 2-undecanone (25, 50, or 100 μM) for 48 h, respectively. After the incubation, the cells were fixed in cold methanol, permeabilized with 0.1% TritonX-100 and blocked with 5% bovine serum albumin. Then, the cells were incubated with a p-H2A.X (1:100) or a Nrf2 (1:100) antibody for 1 h and then stained with a secondary fluorescent antibody (1:200; Alexa Fluor 568, Abcam Inc. Cambridge, MA, USA). Finally, the cells were incubated with DAPI for another 20 min. Fluorescence signals were detected using a Leica TCS SP8 confocal fluorescence microscope (Leica, Germany). The relative fluorescence of p-H2A.X in the cells was analysed by ImageJ software.

Pluronic F127 (P2443) was purchased from Sigma-Aldrich and ropivacaine (R413090) was obtained from Mackline, while Imiquimod (HY-B0180A) and doxorubicin (Dox, HY-15142) were purchased from MCE. Butorphanol (H20020454) was purchased from Hengrui. Anti-mouse CD45-PE-cy7 (103114), anti-mouse CD11c-APC-cy7 (117323), anti-mouse CD3-APC-cy7 (100221), anti-mouse CD8a-APC (100711), anti-mouse CD16/32-TruStain FcX (101320), anti-mouse CD86-APC (105011), anti-mouse CD11b-PE (101207), anti-mouse Gr1-APC (108411), anti-mouse CD4-PE (100408) and anti-mouse Foxp3-AF488(126406) antibodies were purchased from BioLegend. Anti-mouse CD80-FITC (FW0149), anti-mouse MHC-I-APC (FW2919), and anti-Rb Phospho-p38 (Thr180/Tyr182) (DL3485) were purchased from Elabscience. Anti-LC3 (ab192890) and anti-P62 (ab109012) antibodies and Alexa Fluor 568 and Alexa Fluor 488 secondary antibodies were purchased from Abcam. BD™ Cytometric Bead Array (CBA) Mouse TNF Flex Set (558299), Mouse IFN-γ Enhanced Sensitivity Flex Set (562233) and Mouse Enhanced Sensitivity Master Buffer Kit (562246) were purchased from BD Bioscience.

To collect the spinal cords after contusion injury or sham operation mice, mice were anesthetized and then rapidly perfused transcardially with 0.9% saline, followed by 4% PFA. Then, the spinal cords were cryoprotected in 30% sucrose for 3 d at 4°C, before being sectioned. Serial cryostat sections (14 mm thick) were obtained. For immunofluorescent staining, the sectioned slices were permeabilized with 0.3% Triton X-100 in PBS for 30 min and then blocked with 5% BSA for 1 h. After incubation with goat anti-CD31 Alexa Fluor 488-conjugated antibody (1 : 100, R&D, USA), rabbit anti-ki67 (1 : 200, Invitrogen, USA), and rabbit anti-Nrf2 (1 : 200, Proteintech) overnight at 4°C, the sectioned slices were incubated with secondary antibodies anti-rabbit Alexa Fluor 488 or Alexa Fluor 568 (1 : 400, Abcam) for 1.5 h. The slices were mounted with Fluoroshield™ with DAPI (GeneTex Inc.). Signals were analyzed by a fluorescence microscope (Zeiss Apotome 2). Positive cells were quantified using the ImageJ software.
Cell samples were fixed with 4% PFA for 20 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with 5% BSA for 30 min and then incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation for 1.5 h. After washing with PBS, cells were stained with DAPI.