Ab85763

IHC was performed according to Envision two-step method. The microarray was dewaxed by xylene and hydrated by graded alcohol. Tris/EDTA solution (PH = 9.0) was selected as the antigen repair buffer, and hot antigen repair was carried out in a pressure cooker using the high-pressure steam method. The tissue microarray was soaked with 3% H₂O₂ solution for 10 min and then incubated with 10% goat serum for 1 h. The tissue chips and rabbit anti-human JAG1 antibody (1:200, # ab85763, Abcam, USA) were incubated overnight at 4 °C. The tissue chip was incubated with the goat anti-rabbit antibody (1:500, #ab6721, Abcam, USA) at room temperature for 30 min. DAB dye was used for visualization processing, and the tissue chip was counterstained with hematoxylin.
The expression of JAG1 protein was assessed semi-quantitatively through the combination of staining positive intensity and the proportion of staining positive cells^{32 (link)}. The IHC cut-off value was 90 via the X-tile software^{33 (link)}. A score of less than 90 was low or no expression, and the score higher or equal to 90 was a high expression.

RIPA reagent was used to lysate cells and leach total protein. The same amounts of protein samples were separated in SDS-PAGE and then transferred to the PVDF membrane. The membrane was soaked in 5% skim milk at room temperature for 2 h. Rabbit anti-human JAG1 monoclonal antibody (1:1000, # ab85763, Abcam, USA) or GAPDH monoclonal antibody (1:2000, # ab9485, Abcam, USA) was incubated at 4 °C overnight. Finally, the membrane was incubated with a secondary antibody (1:5000, #ab6721, Abcam, USA). The proteins were exposed and photographed in a ChemiDoc XRS⁺ system.

Human third molar samples were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight and then washed twice with distilled water. After decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for more than 3 month, the demineralized tooth samples were dehydrated in an alcohol gradient and embedded in paraffin. Sections of 5 μm thickness were de-waxed and rehydrated followed by antigens retrieval with gastric enzyme. The slides were blocked by 2% BSA and incubated with primary antibodies at 4 °C overnight. The following primary antibodies were diluted in PBS and used: mouse anti-MCAM (1:100) (Abcam ab85763), rabbit anti-JAG1 (1:100) (Abcam ab78479), and rabbit anti-PDGFRa (1:500) (CST AF1062). After washing in PBS, sections were incubated with secondary antibodies for 1 h at room temperature. The following secondary antibodies were diluted in PBS and used: anti-rabbit Alexa Fluor 488 (antgene ANT024), anti-mouse Alexa Fluor 594 (antgene ANT029) and anti-rabbit Alexa Fluor 647 (antgene ANT032). Afterward, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei. Stained samples were imaged with a ZEISS LSM 710 confocal laser-scanning microscope and analyzed by ZEISS ZEN microscope software.