Blockaid

Staining of FFPE tissue slides with enriched libraries was performed on Dako autostainer. After baking slides at 60 °C for 1.5 h, epitope retrieval was done on Dako PT-Linker at pH9, 98 °C, 22 min. The staining on Dako autostainer includes 5 min peroxidase inhibition with 450 µl of solution, containing disodium hydrogenorthophosphate 5% ≤ x < 7%, H₂O₂ 3% ≤ x < 5%, phosphoric acid, monosodium salt, monohydrate 1% ≤ x < 2%, 1 h incubation with 450 µl of binding cocktail (3.4 pmol of enriched library, 0.65 ng/µl sheared salmon sperm DNA, 0.65 ng/µl yeast tRNA, 10% BlockAid (Life Technologies), 30 min incubation with 2 µg/ml of Streptavidin Poly-HRP (Life Technologies), supplemented with 3 mM MgCl₂, 10 min staining with DAB solution, supplemented with 3 mM MgCl₂, followed by 5 min incubation with 450 µl of Hematoxylin (2 ng/µl final conc.). Rinsing with 1× PBS, 3 mM MgCl₂ buffer was done between each step. Finally, the stained slides were dehydrated with ethanol and xylene and covered by coverslip for long-term storage. Microscopy was done on an Olympus BX41.
Histological scores for both nuclear and cytoplasmic staining were calculated as the sum between intensity levels (1, 2 and 3) multiplied by the percentage of the cells with this particular intensity.

PFA fixed coronal 30 μm striatal brain sections were washed 3 × 5 min in 1 x PBS and followed by 1 hr incubation with Blockaid (Life Tech - B10710) at RT with gentle rocking. Rabbit anti-mu opioid receptor (MOR) antibody (1:1000; EMD Millipore: AB5511) was applied at 4 °C in 1 x PBS containing 0.25% Triton-X-100 (PBS-Tx) for 24 hr. Sections were washed with 1 x PBS 5 × 5 min, incubated for 1 hr at RT with a goat anti-rabbit Alexa 488 secondary antibody (1:500, Thermo: A-11008) in PBS-Tx, and washed with cold 1 x PBS 5 × 5 min. Processed sections were mounted on SuperFrost slides (VWR: 48311–703), and coverslipped with Prolong Gold antifade (Life Tech: P36935) mounting media.
Striatal sections containing tdTomato labelled DA axons and patch (striosome) regions visualized by high density MOR signal were imaged on an Olympus FV3000 confocal microscope equipped with 488 and 640 nm lasers and a motorized stage for tile imaging. Z stack images captured the entire thickness of the section at 2.75 μm steps with a 10 x air/0.4 N.A. objective (UPLXAPO10X).
Patch ROIs were manually drawn around the high intensity MOR regions in FIJI (NIH). The same ROIs were then moved outside the high intensity MOR signal to get fluorescence measurements in striatal matrix. Quantification of tdTomato fluorescence intensity signal for patch and matrix ROIs was done on max-projected Z stack images.

Cells grown on Nunc chamber slides were fixed with 4% paraformaldehyde and permeabilized in PBS containing 0.5% Triton X-100 and blocked in Block-Aid (Life Technologies) for 1 h at room temperature. Cells were then incubated with MF20 monoclonal antibody (DSHB) against MHC (1:40 dilution;) overnight at 4 degrees. Secondary antibody Alexa Fluor 488-conjugated secondary antibody (1:200 dilution; Life Technologies) for 1 h at room temperature. Cells were mounted with ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies).