Alexa fluor 488 goat anti rabbit igg

Brain tissue sections were deparaffinized and rehydrated (xylene I 15 min, xylene II 15 min, anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, 85% ethanol 5 min, 75% ethanol 5 min, distilled water washing), and antigen repair was performed in a microwave oven (tissues were placed in a box filled with EDTA antigen repair buffer (pH 8.0), wash with phosphate-buffered saline (PBS) three times (5 min), treated with spontaneous fluorescence quenching agent 5 min, wash 10 min, blocked with bovine serum albumin for 30 min, sections were then incubated overnight with rabbit anti-IL-6 polyclonal antibody (1:300; Servicebio; Cat. No. #GB11117) at 4′C, wash with PBS and subsequently incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:400; Servicebio; Cat. No. #GB25303) for 50 min at 37′C, wash with PBS three times (5 min), then the sections were incubated with DAPI for 10 min at room temperature to stain the cellular nuclei. A fluorescence microscope (Nikon Eclipse C1, Japan) was used to acquire images.

Xiaoyao Pills (XYW) (TaiJi, China), fluoxetine hydrochloride (FLX) (Patheon, France) and Amitriptyline hydrochloride (Sigma, USA) were dissolved in 0.9% saline to prepare solutions. Paeoniflorin (C₂₃H₂₈O₁₁), Liquiritin (C₂₁H₂₂O₉), Glycyrrhizic acid (C₄₂H₆₂O₁₆), Ligustilide (C₁₂H₁₄O₂) (Cheng Du Ai Fa, China). LPS (Escherichia coli 055:B5) was provided by Sigma (St. Louis, MO). Rabbit anti-TrkB antibody (1:1000; Cell Signaling Technology; Cat. No. #4603), rabbit anti-cAMP response element-binding protein (CREB) antibody (1:1000; Cell Signaling Technology; Cat. No. #9197S), rabbit anti-p-CREB antibody (1:1000; Cell Signaling Technology; Cat. No. #9198S), rabbit anti-β-Tubulin antibody (1:1000; Cell Signaling Technology; Cat. No. #2128). Rabbit anti-brain-derived neurotrophic factor (BDNF) antibody (1:1000; Abcam; Cat. No. #ab108319). Rabbit anti-DLG4, PSD95-specific, polyclonal antibody (1:1000; Proteintech; Cat. No. #20665-1-AP) and rabbit anti-synaptophysin polyclonal antibody (1:1000; Proteintech; Cat. No. #17785-1-AP). Rabbit anti-GAPDH antibody (1:1000; Servicebio; Cat. No. #GB11002). Rabbit anti-IL-6 polyclonal antibody (1:300; Servicebio; Cat. No. #GB11117) and Alexa Fluor 488 goat anti-rabbit IgG (1:400; Servicebio; Cat. No. #GB25303).

The effect of sh-PTGS2, RUNX1-shRNA3, or sh-NC control on CRC cells was evaluated by detecting matrix metallopeptidase 9 (MMP9) and PCNA expression with an immunocytochemical method. 24 hh post transfection, HCT116 and SW480 cells were fixed, permeabilized by incubation in 0.5% Triton X-100, and washed in 1 ×  phosphate buffered saline (PBS). Following blocking in 3% BSA (Cat#ST2249-5g, Beyotime) for 30 min, probing was carried out with primary antibody (Servicebio) against PCNA (Cat#GB11010, 1:500 dilution) or MMP9 (Cat#GB11132, 1:1000 dilution) as described^{31 (link)}. The Alexa Fluor 488 goat anti-rabbit IgG (Cat#GB25303, Servicebio) at 1:500 dilution in 1 ×  PBS was used as the secondary antibody. After nuclear staining with DAPI, the cells were visualized on the Olympus fluorescence microscope. Images of three random fields were obtained and the fluorescence intensity was quantified by ImageJ to get an average fluorescence intensity of PCNA or MMP9.