Anti-MAPK immuno staining Membranipora
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:
S1: 3h, 10h, 11h, 13h, 16h | S2: 18h, 22h, 26h, 30h, 42h |
S3: 46h, 50h, 68h, 70h, 88h | S4: 3d, 4d, 5d, 9d |
- Mix samples in PTx.
- Exchange 4x in 30 min.
- 2x 15 min PBT.
- 1x 40 min PTx+NGS.
- Incubate with Anti-MAPK 1:200 in 200 µL of PTx+NGS.
16/07/14
- 3x 5 min PBT.
- 4x 30 min PBT.
- 30 min PTx+NGS.
- 1:250 Anti-Mouse POD (Jackson) + Anti-Mouse HRP in PTx+NGS and incubate overnight in cold room at 21h.
17/07/14
- Washed out secondary with 3x 5min and 4x 30 min PBT.
- Washed 3x 10min in TNT buffer.
- Develop with TSA+Cy5 for 5 min.
- Stopped the reaction in PTx.
- 45 min with 1:4000 Sytox Green.
- 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.