Chemmate dako envision detection kit

To identify the effect of circRNF13 on PC cells in vivo, we managed to construct a PC-bearing nude mouse model with intratumoral injection of circRNF13 siRNA. The tumor size and weight were measured, and IHC was performed as previously described [10 (link)]. Tissues sections were incubated with antibodies against PCNA (CST) or ki67 (CST) at 4°C overnight. ChemMate DAKO EnVision Detection Kit (Dako) was used for immunostaining.

Tumor tissues and organs were fixed with formalin, embedded in paraffin, and sectioned at a 5-mm thickness. The organs were stained with hematoxylin and eosin. The tumor tissue sections were examined by IHC staining by anti-α-SMA (CST), anti-CD4 (Abcam) and anti-CD8α (Abcam). Briefly, the sections were exposed to 3% H₂O₂ in methanol after deparaffinization and rehydration and then blocked with 1% BSA for 30 min at room temperature. After blocking, the sections were incubated with primary antibody overnight at 4 °C, followed by peroxidase-conjugated secondary antibodies (ChemMate DAKO EnVision Detection Kit) and detection reagents. CD4⁺ and CD8⁺ cells were quantified by measuring the number of stained cells in sections from three mice in each group.

Primary OS tumor and healthy tissues were provided by the 920th Hospital of Joint Logistics Support Force of the Chinese People’s Liberation Army, Department of Orthopedics. Protein levels of MIF, CSNK2A2, VDAC2, and ODC1 in both tumor and para-cancerous tissues, were evaluated by IHC. Briefly, the tissues were thawed at room temperature and were subsequently boiled in sodium citrate at 80–90℃ for 25 min and incubated with sodium citrate containing 0.1% H₂O₂ in Tris-HCl for 1.5 h for rehydration and antigen retrieval. Tissue sections were then incubated with 3% H₂O₂ solution for 10 min, followed by incubation with 5% bovine serum albumin (FBS, Sigma, USA). Next, the samples were incubated with primary antibodies against MIF (1:500, Abcam, UK), CSNK2A2 (1:500, Abcam), VDAC2 (1:500, Abcam), and ODC1 (1:500, Abcam) at 4 °C overnight. The samples were then washed three times with Tris washing buffer and probed with biotinylated secondary antibodies (Abcam) and stained according to the instructions provided by the manufacturer of the commercial IHC kit (ThermoFisher Scientific, USA), followed by counterstaining and visualization with the ChemMate DAKO EnVision Detection kit (DAKO) and evaluation under light microscopy (ThermoFisher Scientific) by two experienced pathologists.

Slides of human tissues were obtained from Biochain (AMS Biotechnology, Abingdon, UK). Paraffin embedded sections of human and mouse tissues were deparaffinised, blocked in peroxidise blocking solution for 5 minutes and then washed and blocked in goat serum diluted 1∶5 with PBS for 10 minutes. Primary antibody was added as follows: anti-eEF1Bα from Proteintech (Manchester, UK; 1/100), anti-eEF1Bδ from Proteintech Group (Manchester, UK; 1/400) and anti-eEF1Bγ from Abnova (Taipei, Taiwan; 1/100). The slides were then incubated and visualised using ChemMate DAKO EnVision Detection Kit (DAKO) according to the manufacturer's instructions. In brief, the slides were then washed in PBS and three drops of ChemMate DAKO Envision/HRP Rabbit/Mouse secondary antibody (DAKO Cytomation; Agilent Technologies, Wokingham, UK) were added to each slide and incubated for a further 30 minutes. The slides were washed with PBS, removed from the sequenzer and 0.5 ml of DAB working solution was added to each slide and incubated for 2 minutes. Finally, the slides were washed in dH₂O, counterstained in haemotoxylin, stained with lithium carbonate and dehydrated in absolute ethanol and 75% ethanol, cleared in xylene and mounted in pertex. The entire procedure was performed at room temperature. Sections were viewed by light microscopy on Olympus BX51 using DP software (Olympus).

To compare the expression of MMP1 in other studies, we retrieved two publicly available sets of microarray data (GSE2340024 (link) and GSE2034725 (link)) from the Gene Expression Omnibus (GEO) database. GSE23400 and GSE 20347 consist of gene expression data from 53 and 17 Chinese ESCC patients, respectively. The MMP1 levels in tumors and corresponding normal specimens were compared and T/N ratios were calculated.
Immunohistochemistry (IHC) study was performed on the formalin-fixed paraffin-embedded ESCC tissues according to the manufacturer’s instruction using anti-MMP1 antibody (Merck; MAB-3307; 1:300 dilution) and anti-mouse/rabbit secondary antibody conjugated with HRP (ChemMate DAKO EnVision Detection Kit, Code: K5007, DAKO).