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
  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 ┬ÁL 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.

MAX_Mmem_MAPK_1.lif - Series003 MAX_Mmem_MAPK_1.lif - Series016 MAX_Mmem_MAPK_1.lif - Series022