Rats were euthanized via excess carbon dioxide inhalation, and the coccygeal discs were collected 6 weeks after implantation, and an immunohistochemical analysis was performed for aggrecan and type II collagen. We performed immunofluorescence analysis for a calcitonin gene receptor protein (CGRP), Tie2, brachyury, matrix metalloproteinase-13 (MMP-13), human nuclei antibody, tumor necrosis factor-alpha (TNF-α), and interleukin-1-beta (IL-1β). The harvested tissues were fixed overnight in a 4% paraformaldehyde (PFA) solution and decalcified by using a decalcification solution (RapidCal Immuno; BBC Biochemical, Mount Vernon, WA, USA) for 2 weeks. Then, discs were embedded within paraffin wax and sectioned longitudinally using a microtome (Leica) into sections of 5–10 µm thickness. For the immuno-staining, the first sections were dewaxed, rehydrated, and after that stained with primary antibodies against aggrecan (ab36861; Abcam, Cambridge, UK, 1:1000), type II collagen (ab34712; Abcam, Cambridge, UK, 1:100), CGRP (ab47027; Abcam, Cambridge, UK, 1:200), Tie2 (NBP2-20636; Novus Biologicals, Littleton, CO, USA, 1:200), brachyury (sc-166962; Santa Cruz, Dallas, TX, USA, 1:200), human nuclei antibody (MAB1281; Sigma Aldrich, St. Louis, MO, USA, 1:200), TNF-α (ab6671; Abcam, Cambridge, UK, 1:200), MMP-13 (ab39012; Abcam, Cambridge, UK, 1:200), and IL-1β (AF-501-NA; Novus Biologicals, Littleton, CO, USA, 1:200). Then, after 24 h of incubation, sections were washed with phosphate-buffered saline with Tween 20 and again incubated with the secondary antibody anti-Rb horseradish peroxidase (Roche Diagnostics Ltd., Basel, Switzerland), and Alexa Fluor 488 (A11034; Invitrogen, Waltham, MA, USA, 1:400), Alexa Fluor 488 (A11029; Invitrogen, Waltham, MA, USA, 1:400), Alexa Fluor 568 (A10042; Invitrogen, Waltham, MA, USA, 1:400), and Alexa Fluor 647 (A21469; Invitrogen, Waltham, MA, USA, 1:400)-conjugated secondary antibodies. After that, specimens were carried out for the washing step, then the specimens were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Abcam, Cambridge, UK, 1:1000) and incubated for 10 min. The sections were mounted and finally examined using a fluorescence microscope (Zeiss 880, Oberkochen, Germany, and Leica SP5, Wetzlar, Germany). The percentages of the positive area for aggrecan and type II collagen and positive cell number relative to DAPI for CGRP, Tie 2, brachyury, MMP-13, human nuclei antibody, TNF-α, and IL-1β were calculated using ImageJ software (Version 1.50b, https://imagej.nih.gov/ij/ (accessed on 10 November 2022)).
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