Alexa 488 conjugated anti mouse igg

Mouse brains were removed after cervical dislocation and immediately frozen in powdered dry ice. Coronal sections containing the LGP (Bregma − 0.58 to − 0.94) [40 ] were cut at 20-μm thickness on a cryostat (Leica CM1860, Leica Microsystems, Japan). Eight sections per mouse with 60-μm intervals were mounted on a PPS membrane slide (#11600294; Leica Microsystems). The slides were kept in the cryostat at – 20 °C for 1 h, placed on a hotplate at 40 °C for 35 s, and immediately dried under a hair dryer. Completely dried sections were fixed in ice-cold acetone for 4 min and again dried quickly. To identify GFAP-astrocytes, sections were incubated with GFAP antibody (1:60, MAB360, EMD Millipore, USA) diluted in PBS containing 10% BSA, 1% RNase inhibitor and 4% DTT for 5 min. After washing with PBS, they were incubated with Alexa 488-conjugated anti-mouse IgG (1:100, Jackson ImmunoResearch, USA). Blue-fluorescent Nissl stain (1:50, Neurotrace 435/455, Thermo Fisher Scientific, Japan) was used simultaneously as a counterstain.
We dissected out tdTomato-expressing cells with bushy morphology (Olig2-astrocytes) and Alexa 488-labeled cells (GFAP-astrocytes) from a single section of Olig2^CreER-Ai27 mice using an LMD 6500 system (Leica Microsystems). For each astrocyte sample, about 500 cells were dissected from eight coronal sections per mouse and were subjected to RNA extraction.

Arecoline was purchased from Sigma–Aldrich (St. Louis, MO, USA). N-acetyl cysteine (NAC) and (picropodophyllin) PPP were purchased from Tocris Bioscience (Bristol, UK). Monoclonal mouse anti-human antibodies against α-SMA (1A4), vimentin (9E7E7), and ZEB1 (416A7H10) and rabbit polyclonal anti-human antibody against COL1A1 (H-197) and IGF-1Rβ (C-20) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Rabbit polyclonal anti-human antibodies against p-IGF-1R^Tyr1161 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from GeneTex Inc. (Hsinchu City, Taiwan). A collagen solution from bovine skin was purchased from Sigma–Aldrich. Horseradish peroxidase–conjugated antimouse IgG or anti-rabbit IgG antibodies were purchased from PerkinElmer (Waltham, MA, USA). Alexa 488–conjugated antimouse IgG and Alexa 594–conjugated anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).