Anti gfap z0334

Cells were fixed with a 4% paraformaldehyde (PFA, Electron Microscopy Science), 4% sucrose (Sigma) phosphate buffered saline (PBS, Thermo Fisher) solution. Saturation and permeabilization were performed simultaneously with a 1% bovine serum albumin (BSA, Sigma) 0.2% Triton X-100 (Sigma) PBS solution for 30 min at RT. Antibodies were diluted in 1% BSA PBS solution. Primary antibodies were incubated overnight at 4 °C. C20R antibody from Abcam diluted at 1/1000 was used to stain endogenous aSyn, antibodies 59264 and 51253 from Abcam were used to stain phosphorylated aSyn (pSyn), anti-microtubule associated protein 2 (MAP2) HM-2 clone antibody from Sigma was used at 1/500, anti-ß-III-tubulin (TUJ1) clone SDL.3D10 from Sigma was used at 1/1000, anti-CD68 clone FA-11 from Abcam was used at 1/1000 to stain microglia, and anti-GFAP Z0334 from Dako was used at 1/500 to stain astrocytes. Secondary antibodies coupled to Alexa Fluor 488, 555 or 633 (Thermo Fisher) were used at 1/500 and incubated 1 hour and a half at room temperature. Hoechst probe (Sigma) was incubated at the same time as secondary antibodies, at a concentration of 1/5000. Cells were washed between each step with a 1% BSA PBS solution, and were stored in a 0.1% azide (Sigma) PBS solution for preventing bacterial growth.

Rats were sacrificed through anesthesia 5 days post‐injury and perfused transcardially with PBS and 1% paraformaldehyde (PFA) successively. Fixed the brain tissues in 1% PFA for 12 h and 30% sucrose for 3 days at 4°C, then embedded in optimal cutting temperature compound (OCT), stored at −80°C. Sections were cut at 10‐μm thickness and prepared for immunofluorescence. The primary antibodies used were: anti‐GFAP (Z0334) (DAKO, Hamburg, Germany) and anti‐Iba1 (10904‐1‐AP) (ProteinTech). Secondary antibodies included: Alexa Fluor 488‐conjugated donkey anti rabbit (A‐21206) (Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 555‐conjugated donkey anti rabbit (A‐31572) (Invitrogen). Slices were analyzed via confocal microscopy. Cell numbers of microglia and astrocytes in a HPF (high power field) of 200× view on the focus of lesion were counted artificially.