Rabbit anti txndc9

Briefly, xylene dewaxed and alcohol-rehydrated paraffin sections were placed in a bottle filled with a 0.01 M trisodium citrate solution and microwaved. After heating, slides were thoroughly rinsed in cool running water for 5 min. Sections were immersed in 3% H₂O₂ at room temperature for 30 min to block any endogenous peroxidase activity. Add rabbit anti-TXNDC9 (Abcam, Cambridge, UK), diluted 1:200 at an appropriate concentration to the tissue, and incubate at room temperature for 2 h, or in a moist chamber overnight. Adding fluorescently conjugated secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor 594) (Abcam, Cambridge, UK, ab150080), and incubate for 30 min at room temperature in the dark. Add a small volume of mounting media containing DAPI (Abcam, Cambridge, USA, ab104139) stain and add a coverslip of 15 μl is typically enough to cover a tissue underneath a coverslip fully.

Briefly, paraffin sections deparaffinized with xylene and rehydrated with alcohol were placed in a bottle containing 0.01 M trisodium citrate solution and heated in a microwave. Then sections were rinsed in cold water for 5 min and soaked in 3% H₂O₂ for 30 min at room temperature to block endogenous peroxidase activity. Washed with Tris-buffered saline (TBS) pH 7.4. Then incubated at room temperature for 2 h, or in a moist chamber overnight at 4°C with rabbit anti-TXNDC9 (1:200, Abcam, Cambridge, UK). Sections were then treated with biotin labeled goat anti-rabbit secondary antibodies (Abcam, Cambridge, UK) at 37°C for 30 min, followed by horseradish peroxidase-labeled streptavidin solution. Signals were developed by using diaminobenzidine (DAB) chromogen (Dako, Glostrup, Denmark) as substrate. Meyer's hematoxylin solution (Dako, Glostrup, Denmark) was used as a nuclear neutralizing dye. Incubation without specific antibody was used as a negative control.

The primary antibodies used in this study were rabbit anti-cleaved caspase 3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin (1:3000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-NF-κB p65 (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Lamin B1 (1:1000, Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-TXNDC9 (1:200, Abcam, Cambridge, UK). Species-specific secondary antibodies were purchased from Abcam (Cambridge, UK).