10 μl syringe

Intracerebroventricular injections of AAV were performed as previously described with some minor modifications [7 (link), 28 ]. Briefly, a 32-gauge needle (product #7803–04, 0.5 in. custom length, point style 4, 12 degrees, Hamilton Company) attached to a 10 μl syringe (Hamilton Company) was inserted into the lateral ventricle of cryoanesthetized C57BL/6J pups at postnatal day 0. The needle was inserted at a 30-degree angle from the surface of the head, and held at a depth of approximately two millimeters. Two microliters (1.5E10 genomes/μl) of AAV2/9-(G₄C₂)₂ or AAV2/9-(G₄C₂)₁₄₉ solution was manually injected into each lateral ventricle. Following injection, pups were placed on a heated pad until they recovered from anesthesia, at which time they were placed back into their home cage.

Intracerebroventricular (ICV) injections of AAV were performed as previously described (Chew et al., 2015 (link); Chew et al., 2019 (link)). Briefly, each AAV solution was diluted to a concentration of 1.5 × 10¹⁰ genomes/μl. C57BL/6 J pups at P0 were cryoanesthetized on ice for approximately 3 min or until they exhibited no movement. Two microliters of either AAV2/9-mycATXN3-Q28 or AAV2/9-mycATXN3-Q84 solution was manually injected into each cerebral ventricle using a 32-gauge needle (Hamilton Company) attached to a 10 μl syringe (Hamilton Company). The needle was inserted at a 30-degree angle from the surface of the head at a point approximately two-fifths the distance between the lambda suture and the eye and held at a depth of approximately 2 mm when injecting. After injections, the pups were placed on a heat pad until they completely recovered from anesthesia; they were then returned to their home cages.

The blocking anti-C1q antibody was produced using the variable domain sequences previously described,^{18 (link)} with isotype mouse IgG2b as the control. Five-week-old female mice were anesthetized with 3% isoflurane and placed on a stereotaxic apparatus (Stoelting) for surgery. All injections were performed with a 10-μl syringe (Hamilton Company) with a pulled-glass pipette tip glued to the end of the needle and a syringe pump (Stoelting Quintessential stereotaxic injector). The injection coordinates were 1.25 mm anterior, ±1.82 mm lateral, and 1.75 mm ventral to bregma. One microliter of anti-C1q or isotype control antibody was injected at a rate of 0.5 μl/min. For Nr4a1 lentivirus microinjection, mice were randomly assigned and microinjected bilaterally with either the control or Nr4a1-GFP lentivirus (1 μl of 1 × 10⁹ viral genomes/μl, Hanbio, Shanghai, China) in the hippocampus according to the above coordinates.

The surgery was performed under anesthesia with 2% isoflurane and mice were aligned in a stereotactic frame (KOPF instruments, Tujunga, CA). Briefly, hair over the cheek was clipped, and the skin was aseptically prepared using alternating betadine and alcohol scrubs. Sagittal incisions were made on the skin and masseter muscle to expose the incisor. Two full-thickness defects of 0.3–0.4 mm diameter and 2.5–3.0 mm apart were created on the bone overlying the incisor mesenchyme using a microsurgical drill and a trephine drill bit. A 10 μl syringe (Hamilton Company, Reno, NV) coupled to a glass capillary pulled pipette was inserted into one of the bone defects and calking material was applied around the glass pipette to form an airtight seal. 500 nl 2% CTB-Alexa488 (Molecular Probes: C-34775) was injected into the incisor mesenchyme over a period of 2 min. The glass pipette was kept in the bone defect for 15 min to allow the retrograde tracer to be fully absorbed by the dental pulp. The masseter muscle and cheek skin were then sutured closed. The mice were then subcutaneously injected with Buprenorphine SR (1 mg/kg) and allowed to recover on a heating pad until fully awake. Trigeminal ganglia were harvested up to 72 h after injection.