TITLE: Spatial mapping and contextualization of axon subtypes innervating the long bones of C3H and B6 mice
AUTHORS: Erica Scheller, Alec Beeve, Jennifer Brazill, Madelyn Lorenz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nerves in bone play well-established roles in pain and vasoregulation and have been associated with the progression of skeletal disorders including osteoporosis, fracture, arthritis and tumor metastasis. However, isolation of the region-specific mechanisms underlying these relationships is limited by our lack of precise maps of skeletal innervation. We developed an optimized workflow for imaging of nerve axons in and around the bone, resulting in novel, comprehensive maps of sympathetic adrenergic and sensory peptidergic axons within and around the full length of the femur and tibia in two strains of mice (B6 and C3H). This protocol is intended to serve as a guide during the design, implementation, and interpretation of future neuroskeletal studies and was compiled as a resource for the field as part of the NIH SPARC consortium.</div></div>

[STEPS]
?. [Tissue isolation and fixation]
Fully anesthetize mouse by intraperitoneal (IP) injection of ≥ 100 mg/kg ketamine + 10 mg/kg xylazine. Confirm anesthesia has been established to a surgical plane or greater depth by toe-pinch reflex.
?. [Tissue isolation and fixation]
Post-fix tissues in 10% NBF for 24 h at 4 ˚C.
?. [Frozen sample preparation]
Fully decalcify bones in 20x volume of 14% EDTA (Sigma-Aldrich E5134), pH 7.4 with gentle shaking at 4 ˚C for 10 days, replacing with fresh 14% EDTA, pH 7.4 every 2–3 days.
?. [Immunohistochemistry]
Air dry slides for 30 min at room temperature (RT).
?. [Immunohistochemistry]
Incubate with secondary antibodies (Table 3) in 1% normal donkey serum in TNT buffer for 3 h at RT or 24 h at 4 ˚C.
?. [Immunohistochemistry]
Wash sections 3 x 5 min in TNT buffer.
?. [Imaging]
Capture serial-tiled images through the depth of the section at step size 2.5 µm with a 10x objective on a confocal microscope. For subsequent neural analysis, the resolution should reach at least 1.5 pixels/µm to adequately capture axons of 1 µm in size or larger. A higher resolution is needed to accurately visualize axons smaller than 1 µm. Tissue clearing may be performed to improve visibility in optically dense regions such as the bone marrow.
?. [Tissue isolation and fixation]
Wash tissues for 2 h in diH2O. Store in PBS at 4 ˚C or proceed to sample preparation.
?. [Tissue isolation and fixation]
Carefully remove tibias and femurs keeping most of the surrounding musculature and soft tissue intact to preserve the periosteum.
?. [Frozen sample preparation]
Cryoprotect tissues by infiltrating with 20x volume 30% sucrose in PBS for 24–48 h at 4 ˚C.
?. [Frozen sample preparation]
Embed bones in optimal cutting temperature compound (OCT, Fisher HealthCare 23-730-571) in deep disposable tissue molds (molds come with VWR Mega Cassettes, VWR 18000-286). Orient the bone so that the side touching the short edge of the mold is the side that will be sectioned first. For transverse sections of the tibia or femur, the knee should touch the short edge of the mold. Fill the mold completely with OCT and gently push the tissue with forceps to ensure that the bottom surface of the tissue is level and the tissue is located in the center of the mold lengthwise.
?. [Tissue isolation and fixation]
Perform cardiac perfusion by first slowly injecting 10–20 mL 1x phosphate buffered saline (PBS, Corning 46-013-CM) with a 20 gauge needle through the left ventricle of the heart until liver is cleared of blood.
?. [Frozen sample preparation]
Wash tissues 3 x 30 min in diH2O.
?. [Frozen sample preparation]
For cyrosectioning, in a cryostat with chamber set to -20 ˚C, orient the OCT block on the chuck so that the knee will be sectioned first in a transverse plane. Allow to set until frozen.
?. [Frozen sample preparation]
Use an appropriate cold source to freeze down the tissue in the OCT block relatively quickly and evenly. Store blocks at -80 ˚C or proceed to cryosectioning.
?. [Immunohistochemistry]
Block with 10% donkey serum and permeabilize with 0.3% Triton X-100 in PBS for 1 h at RT.
?. [Immunohistochemistry]
Wash sections 2 x 5 min with 1x PBS.
?. [Immunohistochemistry]
Incubate with primary antibodies (Table 3) in 1% normal donkey serum in TNT buffer (Table 2) overnight at RT in humidified chamber.
?. [Immunohistochemistry]
Wash 3 x 5 min in TNT buffer.
?. [Immunohistochemistry]
Incubate with DAPI (Sigma-Aldrich, D9542) 1:1000 in TNT buffer for 5 min at RT.
?. [Immunohistochemistry]
Wash sections 3 x 5 min in TNT buffer.
?. [Immunohistochemistry]
Coverslip (Fisher Scientific 12-545-M) with Fluoromount-G (Thermo Fisher Scientific 00-4958-02). Air dry and seal with clear nail polish before imaging.
?. [Tissue isolation and fixation]
When the animal is fully anesthetized, open the chest cavity to expose heart and make a small incision in the right atrium to allow free flow of blood.
?. [Tissue isolation and fixation]
Follow by slowly injecting 10 mL 10% neutral buffered formalin (NBF, Fisher Scientific 23-245684) until fixation is achieved as evidenced by tremors in the extremities.
?. [Frozen sample preparation]
Collect section (up to three) on Colorfrost Plus glass slides (Fisher Scientific 12-550-18). Check sections under a light microscope to assess correct plane by bone morphology. Adjust angle of the block holder if necessary to achieve cross-section.
?. [Frozen sample preparation]
Store slides at -80 ˚C or proceed to immunohistochemistry.
?. [Frozen sample preparation]
Mount the chuck and adjust so that the block surface is parallel to the blade. Face the block so that sectioning results in an even layer across the entire surface of the block. Continue until the tissue sample is exposed.
?. [Frozen sample preparation]
Section sample at 50 µm slowly but steadily using the anti-roll plate or a brush to keep the section flat as it comes off the blade.
?. [Frozen sample preparation]
Continue sectioning through the length of the bone.