Cm3000 cryostat

In a separate set of patients, we embedded cryosections from duodenal biopsies in Tissue Tek OCT compound (Sakura USA, Torrance, CA) and used a Leica CM3000 Cryostat (Leica Instruments GmbH, Nussloch, Germany) to cut 5 μm sections, picked up on poly-L-lysine coated slides, and air-dried. In line with immunohistochemical methods from reports available at time of the experiments [16 (link), 17 (link)], we stained the sections with primary antibodies against CD3, IL-18Rα, and Vα7.2, followed by corresponding secondary antibodies conjugated to: AF555, AF488, and Cy5. Antibodies were obtained from Dako (Carpinteria, CA), BioLegend, or LifeTechnologies. We counterstained with DAPI to visualize cell nuclei. We used the Nuance Multispectral Imaging system (CRI, Woburn, MA) to visualize and captured images with a digital camera. We analyzed the images with ImageJ software (US National Institutes of Health, Bethesda, MD). We defined MAIT cells as CD3⁺IL-18Rα⁺Vα7.2⁺ cells, as described previously [16 (link)]. For each patient, a single unblinded operator, using ImageJ software, enumerated both MAIT and CD3+ cells from a single biopsy section at each time point from paired (available at both day 2 and day 30) samples.

Mid-belly muscle sections were cut into 10μm thickness by a Leica CM 3000 cryostat at −23 °C (Leica Microsystems, Wetzlar, Germany) and then they were stored at −80 °C until immunostaining. Frozen muscle cryosection were dried and were encircled with a Dako pen. Slides were chemically permeabilized with PBS containing Triton 0.5% for 15 min and then washed three times with PBS. Slides were blocked with bovine serum albumin 2% (BSA) for 1 h at room temperature and then incubated overnight with a rabbit anti-laminin primary antibody (L9393 Sigma-Aldrich, St. Louis, MO, USA, dilution 1/200) at 4 °C in a moist chamber. Slides were washed three times with PBS and incubated with anti-rabbit IgG Cy3-labeled secondary antibody (111-165-008, Jackson Immunoresearch Labs, West Grove, PA, USA, dilution 1/200) at 37 °C for 45 min. For SUN1/Laminin/DAPI or Nesprin1/Laminin/DAPI IHC, cryosections were labeled with antibodies against SUN1 (bsm-54420R) or Nesprin1 (ab192234) and laminin (L9393) overnight at 4 °C and labeling using the second antibody was performed for 2 h at 37 °C. Secondary antibodies were coupled to FITC or Cy3 (Jackson Immunoresearch Inc). Hoechst staining (1:1000, B2261, Sigma-Aldrich) was used to visualize nuclei and sections were mounted using Fluoromount G medium. Finally, slides were stored at 4 °C until picture acquisition.

For all surgeries, anesthesia was induced with 5% isoflurane and maintained at 2%–3% (wt/vol) for duration of surgery. Polyamide-insulated stainless steel monopolar electrodes (125 μm in diameter, with 0.2 mm of surface exposed) were bilaterally implanted into the anterior pole of NAc (anterior-posterior [AP] 1.4 mm, medial-lateral [ML] ±1.0 mm, dorsal-ventral [DV] –4.5 mm). Anodes were connected to a screw on the skull, and an additional 2 screws were used to secure the implant. DBS was applied for 1 hour using a portable stimulator set to deliver 50 μA current (biphasic pulses, 90-microsecond pulse width). A stimulation frequency of 130 Hz or 50 Hz was used. DBS was applied 24 hours before behavioral testing. These settings were used to approximate a charge density similar to that used with humans, and set below induction of motor effects as previously described (55 (link)). Harvested brains were sectioned coronally (20 μm) on a Leica CM-3000 cryostat (Leica Microsystems) microtome at –20°C and thaw-mounted onto Fisher Scientific Positive Charge glass microscope slides. Slides were post-fixed in 10% formalin vapor, stained with cresyl violet, and then examined at ×10 magnification under a microscope.