Prolong gold anti fade media

Primary antibodies used for immunocytochemistry (ICC) or immunohistochemistry (IHC) included mouse anti-acetylated alpha-tubulin (1:3000 (ICC/IHC); Sigma (cat # T6793; lot # 088K4829)), rabbit anti-Arl13b (1:3000 (IHC); Proteintech (cat # 17711-1-AP; lot # 00017960)), mouse anti-gamma-tubulin (1:2000 (ICC/IHC); Sigma (cat # T6557; lot # 072M4808)), rabbit anti-Gli3C (1:1000 (ICC); gift from S. Scales) [35 (link)], rabbit anti-PCM1 (1:1000 (ICC/IHC); Bethyl Laboratories (cat # A301-150A; lot # A301-150A-1)), rabbit anti-SMO (1:1000 (ICC/IHC); Abcam (cat # ab38686; lot # GR198520)). Cells and sections stained sequentially with mouse antibodies against gamma- and acetylated alpha-tubulin were blocked with anti-mouse Fab fragments (20 μg/ml; Jackson Immunoresearch (cat # 715-007-003; lot # 114326)) as previously described [24 (link)]. Secondary antibodies were species-specific and were conjugated with fluorescent tags (1:400 (ICC/IHC); Jackson Immunoresearch). Stained sections and cells were coverslipped with Prolong Gold antifade media containing DAPI (Invitrogen).

Skin was fixed at room temperature for 30 min in PLP buffer (0.2 M NaH2PO4, 0.2 M Na2HPO4, 0.2 M L-lysine and 0.1 M sodium periodate with 2% paraformaldehyde), washed twice with PBS and incubated for 30 min in 20% sucrose, prior to being embedded, frozen, cut and stained as previously described [48] (link). IFN-γ staining (AlexaFluor647, BD Pharmingen) was performed overnight at 4°C (1∶75 in PBS containing 2.5% [w/v] donkey serum). Anti-keratin-5 and -14 polyclonal antibodies were from Jomar Biosciences; anti-CD4 (RM4-5) from BioLegend; anti-CD8 (53.67) from BD Pharmingen and polyclonal anti-HSV from Dako North America. Slides were mounted with ProLongGold antifade media (Invitrogen), air-dried and viewed using a Zeiss LSM700 confocal microscope and Imaris 7.1 software (Bitplane).

Terminal procedures were performed in deeply anesthetized rats, with the thoracic portion of the phrenic nerve (~1 cm from the DIAm insertion) excised and placed in Trump's fixative (1% glutaraldehyde and 4% formaldehyde in 0.1 M PBS, pH 7.2). These segments were then rinsed in 0.1 M PBS (pH 7.2), followed by 30 min postfix in phosphate‐buffered 1% osmium tetroxide (OsO4). After rinsing in distilled water, samples were stained with 2% uranyl acetate for 15 min at 60°C, rinsed, dehydrated in progressively higher concentrations of ethanol and 100% propylene oxide, and embedded in Spurr's resin.
Following excision of the phrenic nerve, rats were perfused intracardially with a 0.1 M phosphate‐buffered saline (PBS), euthanized via exsanguination, and then perfused with 4% paraformaldehyde in 0.1 M PBS. The C₁–C₆ segment of the spinal cord was then dissected, post‐fixed in 4% paraformaldehyde for 24 h, and then transferred to 25% sucrose in 0.1 M phosphate buffer for 3 days at 4°C. Spinal cord tissue embedded in cryomoulds (VWR), was cut in longitudinal horizontal sections at 70 μm and mounted on Tissue Tack slides (Polysciences) that were pre‐coated with Cell‐Tak adhesive (Becton Dickinson Lab Ware). Cut sections were then mounted in prolong gold anti fade media (Cat# P36934; ThermoFisher), cover‐slipped and stored until imaging, within 3 weeks of processing.

Cells were fixed with cold 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton-X for 5 min, and incubated in PBS containing 10% normal goat or donkey serum for 1 h at room temperature (RT). Antibodies in 5% serum were incubated at 4C overnight. AdMSC markers include Oct3/4 (1:100, Abcam) and Vimentin (1:100, Sigma Aldrich). Following the overnight incubation, cells were washed and incubated with 555-conjugated anti-rabbit or anti-mouse secondary antibodies (1:500, Southern Biotech) and incubated in the dark at RT for 1 h. Slides were incubated in Hoechst stain (ThermoFisher, 1:2000 dilution) for three minutes, then mounted with ProLong Gold Antifade media (ThermoFisher) and cover slipped (Globe Scientific, #1).