Alexa fluor 555 tyramide superboost kit

Immunostaining for N1ICD required tyramide signal amplification, performed using Invitrogen Alexa Fluor 555 Tyramide SuperBoost Kit (B40923). Following primary antibody incubation, sections were incubated with goat anti-rabbit horse radish peroxidase (HRP), then tyramide signal amplification performed for 30 min, following manufacturer’s instructions.

Mouse brains were fixed via immersion in 4% PFA and embedded in paraffin. Next, 7 µm frontal sections were prepared using a HM355S microtome (Thermo, Waltham, MA, USA) and mounted on SuperFrost Plus slides (Epredia, Portsmouth, NH, USA). Antigen retrieval was carried out by boiling the sections in Tris-based solution (Vector Laboratories, Newark, NJ, USA) for 20 min. Sections were blocked with 10% horse serum in PBS containing 0.1% Triton X-100 (0.1% PBTx) for 1 h and incubated with primary antibodies diluted in 5% horse serum in 0.1% PBTx at 4 °C overnight. Sections were incubated with fluorescent secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), diluted in 5% horse serum in 0.1% PBTx at room temperature for 90 min, and stained with DAPI (Invitrogen). Imaging was performed using a TCS SP5 II confocal microscope (Leica Microsystems, Wetzlar, Germany). For biotin labeling, streptavidin–peroxidase (Jackson ImmunoResearch) was added to primary antibodies and Alexa Fluor 555 Tyramide Super Boost Kit (Invitrogen) was used for signal amplification according to manufacturer’s manual. The following primary antibodies were used: guinea pig anti-Bcl11a [26 (link)], rat anti-Bcl11b (ab18465, Abcam, Cambridge, UK), rabbit anti-FLAG (F7425, Sigma, St. Louis, MO, USA), and chicken anti-GFP (ab13970, Abcam).

Immunostaining for N1ICD required tyramide signal amplification, performed using Invitrogen Alexa Fluor 555 Tyramide SuperBoost Kit (B40923). Following primary antibody incubation, sections were incubated with goat anti-rabbit horseradish peroxidase, then tyramide signal amplification was performed for 30 min, following the manufacturer’s instructions.