Ab62341

Spinal cords were recovered and stained as previously described^{60 (link),75 (link)}. Following terminal anesthesia by pentobarbital, mice were perfused transcardially with 10% formalin (Sigma). Spinal cords and brains were removed, post-fixed overnight, transferred to buffered 30% sucrose for 48 h, embedded in O.C.T. Compound (Tissue-Tek, Sakura-Finetek/VWR) and cryostat-sectioned at 30 μm. Serial horizontal sections of spinal cord containing the lesion sites and brain containing the viral injection sites were cut and processed for immunostaining. The following primary antibodies were used: anti-GFAP (DAKO Z0334, 1:1000, free-floating), anti-GAP43 (1:1000, Benowitz lab), anti-Synaptophysin (Synaptic Systems 101004, 1:1000, free-floating), and RFP (1:500, Abcam ab62341, free-floating). BDA tracing was visualized with streptavidin-HRP (1:300, PerkinElmer SAT704A001EA) antibodies plus Cy3-TSA (1:200, PerkinElmer SAT704A001EA). Sections were cover-slipped using Prolong Diamond Antifade Mounting media with DAPI (ThermoFisher) to stain cell nuclei. Each section was imaged on ZEISS LSM 880.

During the preparation of PRP and PRF, high quantities of platelets and leucocytes are in the buffy coat or embedded in the fibrin clot after centrifugation. To determine whether these leucocytes remained in the wound beds until the end of study, we used DsRed-transgenic mini-pigs as blood donors [40 (link)], and then, the PRP, PRF and PP prepared from them were applied to the full-thickness wounds created on non-transgenic littermate recipients with the same blood type. Blood types were confirmed in advance by mixing donor and recipient blood, and in the absence of agglutination, blood types were concluded to be the same. Except for the source of blood, the other procedures are the same as described in the above methods. Skin biopsies of wound beds were obtained on day 14 post-wounding. After adequate fixation and paraffin embedding, histological slices were cut and subjected to immunofluorescence (IF) [48 (link)] and immunohistochemistry (IHC) [49 (link), 50 (link)] staining using rabbit anti-DsRed primary antibody (ab62341; Abcam, Eugene, CA, USA) and FITC-conjugated and peroxidase-conjugated secondary anti-rabbit antibodies (ab97063; Abcam, Eugene, CA, USA), respectively.

The explants were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen, and sagittally sectioned (10 μm in thickness). Immunofluorescence (IF) was performed as previously described56 (link)57 (link). Briefly, the sections were post-fixed in 4% PFA for 40 min, washed with phosphate buffer saline (PBS) and 3% glycine, permealized with 0.03% sodium dodecyl sulfate, blocked with 3% goat serum, and then incubated with a primary antibody of rabbit anti-RFP (1:100, ab62341, abcam, Cambridge, MA, USA) at 4 °C overnight. After washes with PBS, the sections were incubated with an Alexa 594-conjugated goat anti-rabbit secondary antibody (1:1000, ab150092, abcam, Cambridge, MA, USA) for 2 h at RT. Then the sections were thoroughly washed and mounted with the ProLong Gold Antifade with DAPI reagent (Life Technologies, Grand Island, NY, USA). The images were taken under a fluorescence microscope (BX51, Olympus Optical Co. Ltd., Tokyo, Japan) as described above.

Immunofluorescent staining of cells and tumor sections was performed as previously described [70 (link), 71 (link)]. The primary antibodies, employed in this study, were raised against the following antigens: HIF-1α (ab51608; Abcam), FOXC2 (rat anti-mouse FOXC2 antibody, which also cross-reacts with human FOXC2; developed by Dr. Naoyuki Miura, Hamamatsu University School of Medicine, Japan), E-cadherin (61081; BD Biosciences, San Jose, CA, USA), vimentin (MA1-19319; Thermo Fisher Scientific, Asheville, NC, USA), CD31 (ab28364; Abcam), KDR (MA5-15556; Novus, Centennial, CO, USA), RFP (ab62341; Abcam), SV40 large-T antigen (sc-47; Santa Cruz Biotechnology Inc., Dallas, TX, USA). Fluorescently-labeled secondary antibodies were from Thermo Fisher Scientific.