Alexa 568

Coronal sections were prepared on a cryostat at 20 μm and immediately mounted on slides. Slides were maintained at −80°C until ready for staining. Every 10th section was Nissl stained with Cresyl Violet to identify sections corresponding to areas of interest (Franklin and Paxinos, 2013 ).
Immunostaining was done as described previously (Beaudoin et al., 2012 (link)). The following primary antibodies were used mouse anti-bassoon (1:400, ABCAM ab82958), rabbit anti-gephyrin (1:1000, ABCAM ab32206), rabbit anti-N-methyl-d-aspartate (NMDA) receptor subunit 1 (1:1000, Thermo Fisher Scientific PA3-102), and chicken anti-tyrosine hydroxylase (TH) (1:500, ABCAM ab76442). Secondary antibodies used were goat anti-mouse (1:1000 for all; Alexa568, ABCAM ab175473; Alexa488, Fisher A11029; Alexa647, Fisher A21241), goat anti-rabbit (1:1000 for all; Alexa488 ABCAM ab150077; Alexa568, Fisher A11036), and/or goat anti-chicken (1:1000, Alexa405, ABCAM ab175675). Coverslips were mounted with ProLong Gold antifade (Invitrogen).

For staining for AJ proteins, the following monoclonal antibodies (mAbs) were used: anti-β-catenin (CAT-5H10, mouse IgG, Thermo Fisher Scientific, Waltham, MA, USA) and anti-phospho β-catenin (S33/S37/T41) (Rabbit, Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies used were goat anti-mouse IgG conjugated to Alexa Fluor 647 (Abcam, Cambridge, UK) and goat-anti-rabbit IgG conjugated to Alexa 568 (Abcam). The same panel of mAbs was used for Western blotting along with the anti-GAPDH mAb (Rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA). For chemical inhibition of the GSK-3β activity, we used a cell-permeable GSK-3 peptide inhibitor (Millipore Sigma™ Calbiochem™, Steinheim, Germany) at concentrations (0.2–40 μM) that do not affect the cell viability, as previously demonstrated [41 (link)].

TMAs were stained for IL-6, GM-CSF, and CD33⁺S100a9⁺ cells as a tissue-based surrogate of MDSC as published by Ortiz et al., with modifications.^{43 (link)} After rehydration and antigen recall in citrate buffer (Sigma Aldrich, St. Louis, MO, USA), slides were blocked with 5% BSA in PBS and incubated overnight at 4 °C with Abs to IL-6 (mouse 1:200, SantaCruz, Dallas, TX, USA), GM-CSF (1:100, Abcam), or S100a9 (rabbit 1:200, Abcam) and CD33 (mouse 1:100, SantaCruz) diluted in blocking buffer. Control slides were used to ensure that no background from primary or secondary Abs was present. After overnight primary incubation, slides were washed and incubated with secondary Abs to mouse (donkey anti-mouse 1:1000 Alexa 488, Abcam, Cambridge, UK) or rabbit (donkey anti-rabbit 1:1000 Alexa 568, Abcam) for 1 h at room temperature. Slides were stained with 4,6-diamidino-2-phenylindole (DAPI) at 1:5000 and washed, followed by 30-min incubation in 0.1% Sudan Black B in 70% ethanol to quench background fluorescence. Slides were washed with 0.02% Tween-20 in PBS, and coverslips were mounted using Vectashield Hardset (Burlingame, CA, USA). Images were taken using a Leica SP8 confocal microscope at ×20 (IL-6 and GM-CSF) or ×40 (CD33⁺S100a9⁺). Z-stacks were collected of CD33⁺S100a9⁺ cells, with 4 images collected over 3 µm.