PhD notebook

I realized that I don’t have a proper description of all Membranipora stages with the anti-mapk antibody. I prepared 4 wells with mixed stages as following:

1. Mix samples in PTx.
2. Exchange 4x in 30 min.
3. 2x 15 min PBT.
4. 1x 40 min PTx+NGS.
5. Incubate with Anti-MAPK 1:200 in 200 µL of PTx+NGS.

16/07/14

1. 3x 5 min PBT.
2. 4x 30 min PBT.
3. 30 min PTx+NGS.
4. 1:250 Anti-Mouse POD (Jackson) + Anti-Mouse HRP in PTx+NGS and incubate overnight in cold room at 21h.

17/07/14

1. Washed out secondary with 3x 5min and 4x 30 min PBT.
2. Washed 3x 10min in TNT buffer.
3. Develop with TSA+Cy5 for 5 min.
4. Stopped the reaction in PTx.
5. 45 min with 1:4000 Sytox Green.
6. Embedding with TDE.

I realized that sytox staining was fading very quickly under fluorescent scope and decided to add 1:2000 sytox green into TDE and leave it overnight.

18/07/14

Mounted and scanned sample 1 only in the confocal. They look nice, but maybe next time I would either stop the reaction with detergent solution or develop for shorter time, 3 minutes maybe.