BFP FITC

Luciferase:AF647 BFPLuciferase:AF647

• Infections of 293T cells with Lich Generation 3 virus expressing BFP and Luciferase Reporter proteins are shown.

• Luciferase is found throughout the cell but it is mostly concentrated at the cell periphery and in the extensions while BFP is mainly concentrated in what appears to be nuclear region.

• There was no background in FITC channel.

BFP Luciferase:AF647 BFPLuciferase:AF647

• Additional example of BFP and Luciferase expressing cells. • These cells were also negative in FITC channel.• Differential pattern of BFP and Luciferase expression is even more obvious in this example.

• And some more examples..

BFP Luciferase:AF647 BFPLuciferase:AF647

CE39: Vaginal Tissue, Block 3AIVIS negative control tissue

3A

• Many blue cells (that were negative in FITC channel) were found in just a couple of tissue sections from this tissue block

• All of these cells were Luciferase negative

• And were photoactivatable

HM80: Vaginal Tissue, Block 1A

BFP TRITC

Luciferase:AF647DRAQ5

mCherryLuciferase:AF647DRAQ5

• Brief looking at the stained tissue from this animal reviled several mCherry/Luciferase positive cells.

• Imaging at 32%T and 0.2 ms resulted in mCherry readings of ~3,000, TRITCH reading of ~800 and FITC reading of ~200.

• Imaging at 10%T and 0.2 ms resulted in ~3,000 reading for Luciferase.

• Of note DRAQ5 stained nuclei imaged at 50%T and 0.5ms results in ~700 read outs so nuclei are not visible at this image, however due to the very strong expression of Luciferase if we go all the way to 50%T and 0.5ms the signal is too strong.

• Since it appears from our preliminary work on this tissue that there are several mCherry Luciferase cells but no Blue cells we are considering imaging adjacent slides with DAPI instead with DRAQ5 to also get nice nuclear stain for these cells.

BFP TRITC Luciferase:AF647DRAQ5

HM80: Vaginal Tissue, Block 1A