Rabbit anti mcherry

Twenty μm coronal sections were cut at −20 °C in a cryostat, rinsed in 0.1 M PBS and blocked in 0.1 M PBS with 10% normal goat serum and 0.3% Triton X-100. Sections were then incubated overnight at 4 °C with primary antibodies dissolved in blocking solution: rabbit anti-mCherry (Thermo Fisher, 1:100); rabbit anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA, 1:100); rabbit anti-nitric oxide synthase-2 (Santa Cruz, Dallas, TX, USA, 1:100); mouse anti-CD45 (Santa Cruz, 1:100); rabbit anti-Iba1 (Synaptic Systems, Gottingen, Germany, 1:200). The next day, sections were incubated with secondary antibodies for 2 h at room temperature, as follows: Alexa Fluor 488 goat anti-rabbit (Molecular Probes, Eugene, OR, USA), 1:1,000; Alexa Fluor 488 goat anti-mouse (Molecular Probes), 1:1,000. In addition, 10 μM DAPI (Molecular Probes) was added to the secondary antibody solution in order to label cell nuclei.

Lysates from mφs treated with Hoxa3^mCh or mCherry CM were prepared using IP lysis buffer (50 mM Tris-acetate [pH 7.5], 300 mM NaCl, 1 mg/ml BSA, 2% Igepal, 1 mM EDTA, 1× protease inhibitor mixture [Promega], and 1× sodium orthovanadate). Two micrograms of rabbit anti-mCherry (BioVision) was labeled with biotin (DSB-X; Molecular Probes) and incubated with 50 μg protein lysate, together with 0.4 mg prewashed Dynabeads (FlowComp Flexi system; Invitrogen). The mixture was left overnight at 4°C on a rotator before washing and elution with immunoprecipitation wash buffer (0.1% SDS, 500 mM Tris-acetate [pH 7.5], 300 mM NaCl, and 0.5% Igepal) using a magnet (EasySep; StemCell Technologies).
CM from 293T cells transfected with SP-Hoxa3^mCh or SP-mCherry and purified lysate from mφs treated with CM were analyzed by Western blotting using 10% SDS-PAGE and polyvinylidene difluoride membrane, using the Bio-Rad Turbo Transfer System. Blots were probed at 4°C overnight with 0.1 μg rabbit anti-mCherry Ab in a volume of 6 ml and visualized using the Pierce ECL detection system (Thermo Fisher Scientific). Images were generated using the ChemiDoc MP System (Bio-Rad) and Image Lab Software.

Samples in SDS loading buffer were boiled for 5 min and run on 10% or 15% polyacrylamide gels. After transfer to nitrocellulose or PVDF, membranes were blocked in PBST, 5% (w/v) dry milk powder or 2% (w/v) BSA. Primary antibodies were added in blocking buffer overnight: rabbit anti-mCherry (ThermoFisher, PA5–34974, 1:10 000); mouse anti-HP1β (Millipore, MAB-3448, 1:1000); mouse anti-GFP (Santa Cruz, sc-9996, 1:1000 to 1:5000); mouse anti-UHRF1 (Santa Cruz, sc-373750, 1:1000); mouse anti-His-tag (Santa Cruz, sc-57598, 1:500); mouse anti-FLAG (Sigma, F1804, 1:2000). Membranes were washed three times 10 min with PBST and incubated with secondary antibodies (anti-rabbit HRP, Dako, P0399, 1:10 000; anti-mouse HRP, Dako, P0447, 1:10 000 or anti-mouse IRDye 800CW, LI-COR Biosciences, 926-32210, 1:10 000) in blocking buffer for 1 h at room temperature. Membranes were washed three times 10 min with PBST and incubated with ECL substrate for 2 min before imaging on a ChemiDoc (Bio-Rad) or dried and imaged on a LI-COR Odyssey^® CLx.

7.5 μg of mouse anti‐GFP (BioLegend, San Diego, CA, USA) or rabbit anti‐mCherry (Thermo Fisher Scientific) antibody were cross‐linked to 50 μL of magnetic Dynabeads protein G (Thermo Fisher Scientific) with BS3 cross‐linking reagent (Thermo Fisher Scientific) as per manufacturer's instruction. The incubation time was extended to 1 h at 4 °C, and the quenching was extended to 30 min. Lysates (500 μg of protein) were applied to the bead–antibody complex and allowed to incubate with rotation at 4 °C overnight. Beads were washed in triplicate with eight beads volumes of 1× TBS containing 0.1% Tween‐20 (TBS/Tween). The beads were resuspended in 100 μL of TBS/Tween and transferred to a fresh microcentrifuge tube to avoid eluting proteins bound to the tube wall. Immunoprecipitated proteins were released from the antibody bead complex by heating in 30 μL 1× SDS loading buffer and supernatant transferred to a fresh microcentrifuge tube.

Following electrophysiological recordings, the slices were fixed overnight in 4% paraformaldehyde (Electron Microscopy Sciences). The slices were then equilibrated in PBS containing 30% sucrose and frozen in tissue freezing medium (General Data).
To confirm RV labeling in electrophysiologically-identified GCs within the vicinity of transplants of MGE-derived interneurons, we immunostained all the slices for mCherry, eYFP and biocytin. The slices were thawed and transferred to blocking buffer (5% normal goat serum containing 0.3% Triton-X) for 1 h at room temperature (RT). Slices were incubated for 48 h at RT in primary antibody solution in blocking buffer containing: chicken anti-GFP (1:1000, Aves), rabbit anti-mCherry (1:1000, Invitrogen), and streptavidin Alexa Fluor 647 (1:500, Invitrogen; omitted when staining slices that did not contain biocytin-filled cells). The slices were then washed for 1 h in PBS (KPBS, 0.02 M) and transferred into secondary antibody solution containing goat anti-rabbit Alexa Fluor 568 (1:1000, Life Technologies) and goat anti-chicken Alexa Fluor 488 (1:1000, Life Technologies) for 24 h, before a final wash for 1 h. Sections were mounted on Superfrost Plus slides in Prolong Gold with DAPI (Invitrogen) and stored at –20°C in the dark.

Antibody staining was carried out at room temperature using standard protocols. Antigen retrieval to detect epitopes for antibodies was performed by incubating slides in 150 mM Tris–HCl (pH 9) at 70°C for 20 min, then rinsed for 30 min before overnight incubation in a primary antibody prepared with 5% fetal bovine serum (FBS) blocking solution. Primary antibodies used for this study were as follows: mouse anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, sc-25,290), 1:500; rabbit anti-mCherry (Invitrogen, PA5-34974), 1:500, rabbit anti-Bmpr1b (GeneTex, GTX128200). The next day, slides were rinsed thrice with PBS and then incubated for 2 h in secondary antibody, diluted in the same blocking solution. Secondary antibodies used were as follows: Alexa Fluor 647 nm donkey anti-mouse, 1:500; Alexa Fluor 546 nm goat anti-rabbit, 1:500. Following antibody staining, slides were rinsed thrice with PBS and then stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:10000, Sigma-Aldrich, D9542-10MG) in PBS. Finally, sections were mounted in Mowiol (Sigma-Aldrich, cat. Number 81381-250G) using 24 mm/60 mm glass coverslips (ProSciTech). Slides were stored at 4°C.