Anti myd88 and anti α sma antibodies

LX-2 cells were inoculated in 96-well plates (1 × 10³ cells/well). For fluorescence staining, the cells were fixed in 4% formaldehyde for 15 min and permeabilized with 0.2% Triton-X 100 for 10 min at room temperature. The cells were incubated with 2% BSA to block nonspecific binding sites. Then, the cells were incubated with anti-MyD88 and anti-α-SMA antibodies (Abcam, Cambridge, Cambs, UK) followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA).

Paraffin or cryostat sections of liver tissue were prepared as described previously [26 (link)]. The sliced liver paraffin sections were stained with hematoxylin and eosin (H&E) (Zhongshanjinqiao, Beijing, China). To detect hepatic fat accumulation, cryostat liver sections were stained with Oil Red O (Baso, Zhuhai, Guangzhou, CN). For immunohistochemistry (IHC), cryostat sections were incubated with anti-Ki67 antibodies (BD Pharmingen, San Diego, CA, USA) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. For fluorescence staining, cryostat sections were incubated with anti-F4/80, anti-CD11b, anti-Gr1, anti-CD86 (Santa Cruz Biotechnology, TX, USA), anti-CD206 antibodies (BD Pharmingen, San Diego, CA, USA), anti-MyD88 and anti-α-SMA antibodies (Abcam, Cambridge, Cambs, UK) followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA). Sections were evaluated under a micro-scope (DP71, OLYMPUS, Tokyo, Japan) for bright-field and fluorescence microscopy.