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Shut Up And Let Me Go Midi File. • Western blot analysis of extracts from OVKATE, DU 145, and MCF7 cells using VISTA ((D1L2G™) XP ® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). • Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using VISTA (D1L2G™) XP ® Rabbit mAb performed on the Leica® Bond™ Rx.

• Immunohistochemical analysis of paraffin-embedded human breast carcinoma using VISTA (D1L2G™) XP ® Rabbit mAb. • Immunohistochemical analysis of paraffin-embedded human colon carcinoma using VISTA (D1L2G™) XP ® Rabbit mAb. • Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's B-Cell lymphoma using VISTA (D1L2G™) XP ® Rabbit mAb.

• Immunohistochemical analysis of paraffin-embedded OVKATE (left) and MCF7 (right) cell pellets using VISTA (D1L2G™) XP ® Rabbit mAb. • Immunohistochemical analysis of paraffin-embedded human kidney using VISTA (D1L2G™) XP ® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

• Immunohistochemical analysis of paraffin-embedded human tonsil using VISTA (D1L2G™) XP ® Rabbit mAb. • Flow cytometric analysis of human whole blood cells stained with VISTA (D1L2G™) XP ® Rabbit mAb and co-stained with CD19-PE and CD14-APC. VISTA is present on CD14+ monocytes (right; upper right quadrant) and shows a significant increase in intensity when compared to its isotype control (middle; upper left quadrant). CD19+ B cells are negative for VISTA (left; upper left quadrant).

Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor ® 488 Conjugate) #4412 was used as a secondary antibody. Western Blotting Protocol For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween ® 20 at 4°C with gentle shaking, overnight. NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution. Solutions and Reagents From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: Western Blotting Application Solutions Kit NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

• 20X Phosphate Buffered Saline (PBS): () To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2O, mix. • 10X Tris Buffered Saline (TBS): () To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH 2O, mix. • 1X SDS Sample Buffer: Blue Loading Pack () or Red Loading Pack () Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH 2O. • 10X Tris-Glycine SDS Running Buffer: () To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH 2O, mix. • 10X Tris-Glycine Transfer Buffer: () To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH 2O, mix. • 10X Tris Buffered Saline with Tween ® 20 (TBST): () To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH 2O, mix.