Ab183032

Cells were fixed in 4% paraformaldehyde for 15 min, followed by three washes of DPBS, blocked and permeabilized in PBS containing 0.1–0.2% Triton X-100 and 10% horse serum. The coverslips were incubated with primary antibodies; for NPCs: rabbit anti-PAX6 (CST, mAb#60433, 1:250) and mouse anti-NESTIN (CST, mAb#33475,1:2000); for Neurons: chicken anti-MAP2 (Abcam, ab92434, 1:500), rabbit anti-TBR1 (Abcam, ab183032, 1:250), rabbit anti-VGLuT1 (Abcam, ab227805, 1:500) and Mouse anti-GABA (Abcam, ab86186, 1:400) in the blocking solution overnight at 4 °C. On the next day, they were washed in DPBS and incubated with DAPI (Abcam, ab228549, 1:1000) and corresponding secondary antibodies (Abcam, ab150084, ab150117, ab175711, 1:250) for 60 min at room temperature. Then the coverslips were washed three times, mounted on glass slides using Fluromount-G (mounting medium), and dried overnight while being protected from light. Fluorescence signals were detected using a Leica THUNDER imager and analyzed using ImageJ and MATLAB.

PBS-perfused brains were fixed in 4% paraformaldehyde, and coronal sections were prepared for IFA (thickness: E18.5, 20 μm; postnatal, 30 μm) or IHC and histochemistry (thickness: 3 μm). Mouse monoclonal antibodies against MCMV IE1 (51 (link)); rabbit monoclonal antibodies against SOX2 (Abcam, ab97959), Tbr1 (Abcam, ab31940, ab183032), Ctip2 (Abcam, ab240636), GFAP (Proteintech, 16825-1-AP), Iba1 (Abcam, ab178847), GSDMD (Affinity, AF4012), or CC3 (Cell Signaling Technology, 9661); and rat monoclonal antibodies against BrdU (Abcam, ab6326) or Ctip2 (Abcam, ab18465) were used in IFA or IHC as indicated. Secondary antibodies for IFA included Alexa Fluor 488 goat anti–mouse IgG1 (Invitrogen, A-21121), Alexa Fluor 568 donkey anti–rabbit IgG (H+L) (Invitrogen, A-10042), Alexa Fluor 647 goat anti–mouse IgG1 (Invitrogen, A-21240), and Alexa Fluor 647 goat anti–rat IgG (H+L) (Invitrogen, A-21247). DAPI (Life Technologies) was used for nuclei counterstaining. Horseradish peroxidase–conjugated antibody (Proteintech, KIHC-5) was used for IHC. IFA, IHC, and histochemistry were performed as described previously (52 (link)).

Cells were washed three times with sterile PBS and then fixed using 4% PFA for 20 min. After washing, cells were blocked 0.3% Triton-X and 1% goat serum in PBS for 1 h. Primary antibodies specific for Synapsin1 (1:500; ab254349; Rabbit; Abcam, Cambridge, MA, USA), NeuN (1:500; ab104225; Rabbit; Abcam, Cambridge, MA, USA), Beta-III Tubulin (1:500; MAB1637, Mouse; Kenilworth, NJ, USA), MAP2 (1:1000; Chicken; ab5392; Abcam, Cambridge, MA, USA), TBR1 (1:200; ab183032; Rabbit; Abcam, Cambridge, MA, USA), GFAP (1:500; ab4674; Chicken; Abcam, Cambridge, MA, USA), and Ki67 (1:500; ab245113; Mouse; Abcam, Cambridge, MA, USA) were incubated overnight. After washing, secondary antibodies (chicken 555, rabbit 488, mouse 647; Abcam, Cambridge, MA, USA) were incubated for 2 h. This was followed by 10 min of DAPI Staining Solution in PBS (1:1000, ab228549, Abcam, Cambridge, MA, USA) after which point slides were cover-slipped with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) mounting media and allowed to dry for 48 h.