Anti-MAPK immuno staining Membranipora 

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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.


  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.


  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.


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