Fast blue

3-month old Chat-ires-Cre mice or Chat-tau:eGFP mice were anesthetized and stereotaxically injected (Kopf) bilaterally (200nL per site) in the lateral EC (−4.0mm A/P, ±4.5mm D/V, −3.5mm M/L, empirically determined using landmarks). Viruses: CAV2-DIOhM4Di. mCherry was obtained from Dr. EJ Kremer (Institut de Génétique Moléculaire de Montpellier, France) and AAV9-PCaMKIIa-GCaMP6f.WPRE.SV40 (obtained from U.Penn Vector Core) to visualize the injection site.
Retrograde tracing experiments were conducted via injection with Fast Blue (3% solution in sterile water w/v, Polysciences Inc). 80nL of 3% Fast Blue was injected into the lateral entorhinal cortex of WT animals bilaterally. Mice were euthanized 4 days after injection.

Animals were anaesthetized with ketamine (n = 3) [0.6 ml sterile saline, 0.1 ml medetomidine hydrochloride (1 mg/ml), 0.24 ml ketamine (0.3 mg/ml)], with 250 μL injected interperitoneally per 100 g of rat. 25 μL of Fast Blue (2% w/v in dH₂O) (Polysciences Europe GmbH) was injected in the knee. The animals were bought back to consciousness with the reversing agent atipamezole (5 mg/ml), where 25 μL was injected subcutaneously per 100 g of animal.

Animals were anesthetized with sodium pentobarbital (60 mg/kg) injected intraperitoneally and kept under anesthesia using 1.5% isoflurane. A midline laparotomy was performed to gain access to the urinary bladder. Fast blue (Polysciences Inc., Warrington, PA, USA; 1.5% w/v in water) was injected into the urinary bladder wall using a Hamilton syringe at 6–8 sites (Hamilton Company, Reno, NV, USA). Incisions were sutured in layers under sterile conditions and rats were allowed to recover on a warm blanket and monitored for signs of pain or discomfort. Buprenorphine (2.0 mg/kg) was injected subcutaneously for pain management.

Motoneurons innervating the lateral gastrocnemius (LG) muscle were pre-labeled by injecting 5 µl of 0.1% cholera toxin subunit b conjugated to an Alexa Fluor 555 (CTb-555) (Invitrogen) or 1.5% of Fast Blue (Polysciences) directly into the LG muscle with a Hamilton syringe. Buprenorphine (0.05–0.10 mg/kg) was injected subcutaneously (s.c.) for postsurgical pain management. Retrograde labeling was done one week prior to any nerve injury and under isoflurane anesthesia (induction: 4–5%; maintenance: 2–3%, both in 100% O₂). CTb-555 animals were used for dual color two-photon imaging with CX3CR1-GFP microglia. Fast Blue animals were used for histological processing or to confirm the location of axotomized motoneurons with epi-fluorescence prior to two photon imaging of CX3CR1-GFP microglia.

CD68 expression and cell morphology were also analyzed in microglia ipsilateral and contralateral to the injury at different post-injury times and from animals perfusion-fixed with 4% paraformaldehyde and processed for routine histological immunolabeling. In this case, LG motoneurons were retrogradely labeled with Fast Blue (Polysciences) and the nerve injury performed 1 week after. At 3, 7 and 14 days after the nerve injury, the animals were deeply anesthetized with Euthasol (100 mg/kg) and transcardially perfused first with vascular rinse containing heparin followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The spinal cords were post-fixed overnight in the same fixative solution at 4 °C. The following day they were cryoprotected in 30% sucrose at 4 °C. Transverse 50 µm thick sections were obtained on a freezing sliding microtome and processed free floating. We combined a chicken polyclonal antibody against GFP (Serotect, #obt1644, RRID:AB_620519) with the FA-11 monoclonal antibody (anti-CD68). Primary antibody incubations were done overnight at room temperature. GFP immunoreactivity was revealed with FITC-conjugated anti-chicken IgY antibodies and FA-11 reactivity with Cy3 conjugated anti-rat IgG antibodies. Both secondary antibodies were generated in donkey (Jackson ImmunoResearch Labs).

Motor neurons innervating specific muscles were retrogradely labeled using the tracer FastBlue (Polysciences Inc. #17740) at 5% w/v. Mice were anesthetized using isofluorane and a small incision was performed on the skin to expose the muscle of interest. Tracer was loaded into a Hamilton syringe (Hamilton #1701N) and mounted onto a syringe pump (KD Scientific #78-0220). Tracer was delivered at a rate of 1 µl/min, 1 µl per site, 2–4 sites per muscle, depending on muscle size. Tibialis anterior (TA) was injected at 3 sites per side, soleus at 2 sites per side, lumbar extensors of the spine^{43 (link)} (axial) at 4 sites per side, hindlimb footpads (digit) at 3 sites per side. Phrenic MNs were labeled by transthoracic injection, as previously described^{64 (link)}. The tissue was fixed and harvested 3 days post-injection.