Tris based solution

Following euthanasia, tissues were immediately fixed using 10% formalin for 3 days and processed in the Histology Core Facility of LSU-SVM. For IHC, 5-μm thick paraffin sections were deparaffinized in xylene and rehydrated through graded alcohols. Antigen retrieval was performed by incubating the slides in boiling Tris-based solution (Vector Laboratories) for 1 h. Endogenous peroxidases were inactivated using 3% H₂O₂ for 10 min. Slides were then blocked with 2.5% Normal Horse Serum (Vector Laboratories, United States) and incubated with 1/100 diluted rabbit anti-mouse CD3 (Abcam). After overnight incubation, slides were incubated with goat anti-rabbit IgG (Vector Laboratories) followed by horse anti-goat horseradish peroxidase (HRP, Vector Laboratories). Slides were developed with Vector VIP substrate (Vector Laboratories) and scanned using the NanoZoomer Slide scanner (Hamamatsu Photonics, Japan).

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).