Primary cilia are highly oriented with respect to collagen direction and long axis of extensor tendon.

Tissue Collection

Bilateral digital extensor tendons were collected from 3-week-old male and female Sprague-Dawley rats euthanized by pentobarbital overdose under an IACUC-approved protocol. Tissues were collected from 10 rats for analysis of ciliary incidence and 16 rats for analysis of ciliary orientation. Eight of the rats designated for the incidence analysis were administered bromodeoxyuridine (BrdU) (IP, 25 mg/kg) 2 h prior to euthanasia to label S-phase nuclei, and 2 were injected with saline as non-BrdU controls. 22 Immunohistochemistry (IHC) Primary cilia, tenocyte nuclei, and collagen were characterized in immunofluorescently labeled tendon. Paraffin sections were used for the incidence analysis to ensure antibody penetration throughout the tissue, and whole-mount specimens were used for the orientation analysis to preserve the native tendon ECM. For the incidence analysis, digital extensor tendons were fixed in 10% formalin, embedded in paraffin, and cut into 5-μm-thick sections. One set of sections was immunolabeled for acetylated α-tubulin; a second set was immunolabeled for BrdU; and a third set was double-labeled for acetylated α-tubulin and BrdU. For the single-label specimens, sections were incubated in anti-acetylated α-tubulin or anti-BrdU primary antibodies and then in Alexa 488 or Alexa 568 secondary antibodies, respectively. For the double-labeled sections, following overnight incubation with the acetylated α-tubulin primary antibody, acid denaturation, and neutralization, the sections were incubated in the anti-BrdU primary antibody. The sections were then serially incubated in Alexa 488 and Alexa 568 secondary antibodies. Finally, all sections were coverslipped in a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). Details of these IHC protocols are given in the Supplemental Material . For the orientation analysis, whole fascicles (∼40 μm thick) were fixed in methanol at 4°C for 2 h, permeabilized, and immunolabeled with a mouse anti-acetylated α-tubulin primary antibody and a fluorescent Alexa Fluor 568 secondary antibody to localize primary cilia as described previously. 23 The fascicles were stained with Hoechst 33258 for nuclear visualization. Details of the whole-mount IHC protocol are given in the Supplemental Material .

Multiphoton Microscopy

(MPM) and Image Analysis All specimens were imaged with MPM. In this technique, a fast-pulsed laser raster scanned within the tissue excites both multiphoton fluorescence from exogenous fluorophores and endogenous second harmonic generation (SHG) from arrays of collagen molecules. 24 The MPM system, described previously, included a Ti:Sapphire laser tuned to 800 nm. 25 For the single-label specimens, three signals were collected by detectors equipped with filters corresponding to UV, blue, and visible emissions: SHG from the collagen (400 nm), blue fluorescence from the Hoechst-stained nuclei, and yellow fluorescence from the Alexa 568-labeled primary cilia. 23 For the double-label specimens, three signals were collected by detectors equipped with filters for blue, green, and red emissions: SHG, blue-green fluorescence from the Alexa 488-labeled primary cilia, and yellow fluorescence from the Alexa 568-labeled BrdU. In addition, blue fluorescence from the DAPI-stained nuclei was collected in both the blue and green channels. The laser was focused into the tissue in 1-μm steps, generating z-stacks that were deconvolved and reconstructed for 3D visualization ( Fig. 1a ). Primary cilia and their associated nuclei were tracked throughout the stack. For the incidence analysis, one stack spanning the entire section thickness was collected from each of two sections for each animal. The stacks were analyzed to determine the ratios of a) nuclei with cilia to total nuclei and b) BrdU-labeled nuclei to total nuclei. For the orientation analysis, one image stack spanning the whole fascicle thickness (∼40 μm) was analyzed for each animal using a custom image-processing program in which nuclei and cilia were modeled as ellipsoids and lines, respectively. 26 Outcome parameters included the ciliary length in 3D, the in-plane angle θ with respect to the proximal-distal (y) axis, and the elevation angle ϕ with respect to the cranial-caudal (z) axis ( Fig. 1b ). Details of the imaging are given in the Supplemental Material .

Supplementary Material Supp Fig 1