Guinea pig anti iba1

For single and double immunostaining, the following primary antibodies were used: mouse-anti-GFAP (1:2000, Synaptic Systems, Cat. No: 173011), guinea pig-anti-Iba1 (1:500, Synaptic Systems, Cat. No: 234013), goat-anti-DGL-α (1:200, Frontier Institute, Cat. No: MSFR101310), guinea pig anti-CB1 (diluted 1:200; Frontier Institute, Cat. No: CB1-GP-Af530-1) or rabbit-anti-COX-2 (1:200, Synaptic Systems, Cat. No: 12282). The specificity of primary antibodies has been extensively tested in earlier studies (Hegyi et al., 2009 (link), 2012 (link); Chopra et al., 2019 (link)).

Mice were perfused with phosphate-buffered saline (PBS) and then 4% paraformaldehyde (PFA) in PBS. The brain was dissected and postfixed overnight in 4% PFA-PBS. Mouse brains were soaked in 30% sucrose-PBS for cryoprotection. Brains were sectioned into 40 μm thick slices using a sliding microtome (Leica). Brain sections were blocked in 0.3% Triton X-100 and 10% normal horse serum before overnight incubation in primary antibody at 4 °C. The following primary antibodies were used: guinea pig anti-Iba1 (1:500; Synaptic System, Göttingen, Germany, #234308), goat anti-SOX9 (1:2000; R&D Systems, Minneapolis, MN, USA, #AF3075), mouse anti-GFAP (1:500; Cell Signaling Technology, Danvers, MA, USA, #3670S), mouse anti-Olig2 (1:250; Millipore, Burlington, MA, USA, #MABN50), rabbit anti-ASPA (1:1500; GeneTex, Irvine, CA, USA, #GTX113389), chicken anti-GFP (1:2000; abcam, Cambridge, UK, #13970), and rabbit anti-DsRed (1:1000; TaKaRa, San Jose, CA, USA, #632496). The brain sections were washed three times with PBS before incubation in secondary antibody for 1 h at room temperature. The brain sections were then counterstained with DAPI, mounted onto microscope slides, and covered with mounting media.

Double or triple label experiments were performed with the same basic IsoB4 staining protocol. For combined IsoB4 histochemistry and immunohistochemistry experiments, the following primary antibodies were added at 1:500 dilution in separate sets to the primary label solution containing IsoB4: guinea pig anti-Iba1 (Synaptic Systems, Goettingen, Germany; Cat. No. 234 004); guinea pig anti-NeuN (Millipore Sigma, Burlington, NJ, USA; Cat. No. ABN90PMI), or rabbit anti-S100β (Immunostar, Hudson, NY, USA; Cat. No. 22520). Appropriate Alexa Fluor dye-conjugated secondary antibodies raised in goat were added to the secondary label solution containing Alexa Fluor dye-conjugated streptavidin at 1:500 dilution. For combined IsoB4 histochemistry, immunohistochemistry and EdU histochemistry experiments, Edu staining was performed at the end of the combined IsoB4 histochemistry and immunohistochemistry procedure using the Click-iT EdU imaging kit (Thermo Fisher Scientific, Waltham, MA, USA) with Alexa Fluor-conjugated hydrazide according to manufacturer’s instructions.