Goscript reverse transcription kit

Total RNA was isolated from ankle joint tissues using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s protocol. The RNA was reverse-transcribed into cDNA using the GoScript Reverse Transcription kit (Thermo, USA) according to the manufacturer’s protocol. The mRNA levels were quantified by RT-qPCR using an SYBR Green qPCR Master Mix (Takara, Dalian, China) for 40 cycles on the StepOnePlus RT-PCR system (Applied Biosystems). The primer sequences used to amplify mRNA were shown in Table 1. The levels of mRNA expression were normalized to the reference gene (GAPDH).Table 1

Sequences of Primers Used in This Research

Gene	Forward Primer	Reverse Primer
CAT	5ʹ-CCTGCAACGTTCTGTAAGGC-3’	5ʹ-ATATCAGGTTTCTGCGCGGC-3’
GPx-1	5ʹ-CTCATGACCGACCCCAAGTT-3’	5ʹ-GTCAGAAAGCGACGGCTGTA-3’
IL-6	5ʹ-TGTATGAACAACGATGATGCAC-3’	5ʹ-CTGGCTTTGTCTTTCTTGTT-3’
TNF-α	5ʹ-CTTCTGTCTACTGAACTTCGGG-3’	5ʹ-CAGGCTTGTCACTCGAATTTTG-3’
GAPDH	5ʹ-TCGGAGTGAACGGATTTGGC-3’	5ʹ-TGACAAGCTTCCCGTTCTCC-3’

Rabbit polyclonal anti-Apollon antibodies were purchased from Abcam (cat. nos. ab84429 and ab19609; Cambridge, UK); the PV-6001 two-step immunohistochemistry kit (cat. no. PV-6001) was from OriGene Technologies, Inc. (Beijing, China); Invitrogen^® TRIzol reagent (cat. no. 15596026) was from Thermo Fisher Scientific, Inc. (Waltham, MA, USA); the GoScript Reverse Transcription kit (cat. no. A5001) was from Promega Corporation (Madison, WI, USA); the GoTaq RT-qPCR kit (cat. no. A6001) was from Promega Corporation; paraffin-embedded machines, the paraffin slicing machine and the automatic upright microscope system (DM5000 B) were from Leica Microsystems (GmbH, Wetzlar, Germany); the 400W UV imaging system was from Kodak (Kodak, Tokyo, Japan); and the ABI PRISM 7500 PCR applications were purchased from Thermo Fisher Scientific, Inc.