Goat anti rabbit alexa fluor 594

At 65 ± 2 days postinjection, the AAV-FMRP- and AAV-EV-injected mice were anesthetized using a ketamine–xylazine solution, then transcardially perfused with PBS, followed by 4% paraformaldehyde in PBS (pH 7.4). Serial coronal sections were cut at a thickness of 25 μm using a cryostat (Leica Microsystems, Wetzlar, Germany) as previously described (37 (link),55 (link)). Free-floating sections were washed with PBS, and antigen retrieval was performed as described elsewhere (56 (link)). Monoclonal mouse anti-FMRP 5c2 (57 (link)) was used along with monoclonal rabbit anti-MeCP2 D4F3 (Cell Signaling, Beverly, USA) at a 1:1000 dilution. To confirm the neuronal lineage of the cells, anti-NeuN (MAB377 from Millipore and ab177487 from Abcam) and anti-S100β (ab868 from Abcam and S2532 from Sigma) were used at 1:1000 dilutions to label neurons and astrocytes, respectively. The sections were labeled with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 (1:4000; Jackson Immunoresearch Laboratories, West Grove, USA). DAPI (Sigma-Aldrich) was used to label nuclei. Cell populations quantification was implemented in MATLAB version R2017a (9.2.0) and the code was included in the supplementary information. Only cells fully within this focal plane were quantified. Linear regression was performed using Graphpad Prism 5 to calculate the MeCP2/FMRP correlation parameters.