Alexa fluor 488 goat anti rat igg

Sections were treated for 30 min at room temperature (RT) with a blocking reagent consisting of 2% (v/v) foetal calf serum and 2% (w/v) bovine serum albumin in PBS (pH 7.2). Next, sections were incubated at RT for at least 1.5 h with specific primary monoclonal antibodies (Table 1), diluted 1:20 in a blocking reagent, rinsed 3×10 min with the blocking reagent and incubated at RT for at least 1.5 h with AlexaFluor 488 goat anti-rat IgG (Jackson Immuno-Research Laboratories) diluted 1:100 in the blocking reagent and used as the secondary antibody. After several washes with the blocking reagent and PBS, the sections were stained with 0.01% (w/v) fluorescent brightener 28 (FB28) (Sigma-Aldrich) in PBS for 5 min, then with 2 μg/ml DAPI in PBS (5 min at RT) after which they were thoroughly rinsed with PBS and sterile distilled water. Dried slides were mounted in a Fluoromount (Sigma-Aldrich) anti-fading medium. Negative controls were performed for each antibody used by omitting the primary antibodies. All images were taken using a Zeiss Axio Imager Z2 microscope equipped with an AxioCam Mrm monochromatic camera (Zeiss) with the corresponding software and narrow band filters for AlexaFluor 488 and DAPI.

Primary antibodies used in this study include: rabbit anti-Brn2 (Santa Cruz, 1:50), rabbit anti-Ctip2 (Abcam, 1:1000), rabbit anti-Tbr1 (Proteintech Group, 1:500), rat anti-BrdU (Abcam, 1:1000), rabbit anti-Ki67 (Abcam, 1:500), mouse anti-Pax6 (Cell Signaling Technology, 1:200), rabbit anti-Pax6 (Proteintech Group, 1:1000). Secondary antibodies used include: Alexa Fluor 555 donkey anti-rabbit IgG (Invitrogen, 1:300), Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, 1:300), Alexa Fluor 488 goat anti-mouse IgG (Proteintech Group, 1:300), Alexa Fluor 488 goat anti-rat IgG (Jackson Immuno Research, 1:300). Cell apoptosis was detected by TUNEL FITC Apoptosis Detection Kit (Vazyme, A111). Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Cayman Chemical).