Vectashield plus antifade dapi

Five-micron-thick paraffin sections were prepared. Sections were deparaffinized with xylene and rehydrated with graded ethanol washes. Sections were then cooked in Tris-based antigen-unmasking solution to retrieve the epitopes. Following antigen retrieval, sections were covered with TrueBlack (Biotium, 23007) for 1 min and washed twice with PBS for 10 min. Blocking was done at room temperature for 1 h in 3% donkey serum and M.O.M. blocking reagent (Vector labs). Slides were incubated with primary antibodies raised against DLK1 (1:200; Abcam, ab119930) and CD31 (1:200; Abcam, ab28364) for 48 h at 4 °C. Slides were washed three times with PBS and incubated with corresponding secondary antibodies (anti-mouse Alexa Fluor 546, A10036; anti-rabbit Alexa Fluor 488, SA5-10038; 1:200) for 2 h followed by three washes in PBS. Sections were mounted using VECTASHIELD mounting medium containing DAPI (Vectashield plus Antifade DAPI, Vector lab, H-2000). Images were acquired using a Leica Thunder microscope. The exposure settings and laser gain were kept constant for each condition, and analysis was performed using NIH ImageJ software.

Five-micron-thick paraffin sections were prepared. Sections were deparaffinized with xylene and rehydrated with graded ethanol washes. Sections were then cooked in Tris-based antigen-unmasking solution to retrieve the epitopes. Following antigen retrieval, sections were covered with TrueBlack (Biotium, 23007) for 1 min and washed twice with PBS for 10 min. Blocking was done at room temperature for 1 h in 3% donkey serum and M.O.M. blocking reagent (Vector labs). Slides were incubated with primary antibodies raised against DLK1 (1:200; Abcam, ab119930) and CD31 (1:200; Abcam, ab28364) for 48 h at 4 °C. Slides were washed three times with PBS and incubated with corresponding secondary antibodies (anti-mouse Alexa Fluor 546, A10036; anti-rabbit Alexa Fluor 488, SA5-10038; 1:200) for 2 h followed by three washes in PBS. Sections were mounted using VECTASHIELD mounting medium containing DAPI (Vectashield plus Antifade DAPI, Vector lab, H-2000). Images were acquired using a Leica Thunder microscope. The exposure settings and laser gain were kept constant for each condition, and analysis was performed using NIH ImageJ software.