PhD notebook

Updates from August, 2015 Toggle Comment Threads | Keyboard Shortcuts

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  Bruno Vellutini 16:37 on 2015/08/25 Permalink
  Tags: confocal ( 23 ), Membranipora membranacea ( 140 )   

  Membranipora simple staining 

  Just a simple staining of mixed stages of Membranipora using DAPI and Phalloidin 647 for morphology.

  • 3x 30min 0.2% PTx washes.
  • 1x PTx overnight at 4°C.

  26/08/15

  • Staining with Phalloidin and DAPI for 2h.
  • Let embryos in PBS+DAPI 1:1000 at 4°C.

  It failed… weird.

   
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  Bruno Vellutini 12:09 on 2015/08/25 Permalink
  Tags: mapk ( 26 ), Membranipora membranacea ( 140 )   

  Membranipora in situ + mapk, a re-try 

  Since I saved some de-glycerolled embryos I’ll repeat the immuno staining with a longer developing time.

  gata b

  nk2.1

  • Washed 1h in PBT (4x).
  • Washed 3h in 5% NGS (3x).
  • Added anti-mapk antibody 1:100.
  • Let overnight at 4°C.

  26/08/15

  • Washed primary antibody with 3x PBT 5min + 3x 30min.
  • 5% NGS 3x 30 min.
  • Added both anti-mouse-pod/hrp at 1:250 for 4h at room temp.
  • Washed 4x 5min with PBT and let overnight at 4°C.

  27/08/15

  • Wash 1x PBT.
  • Wash 3x TNT buffer.
  • Develop with cy3 for 5min.
  • Wash with PBS (no detergent solution).
  • Embed in TDE.
   
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  Bruno Vellutini 11:47 on 2015/08/18 Permalink
  Tags: confocal ( 23 ), even-skipped ( 4 ), foxa ( 5 ), gata, mapk ( 26 ), Membranipora membranacea ( 140 ), nk2.1, six3/6 ( 6 )   

  Membranipora MAPK staining on in situ embryos 

  Selected 5 genes for a MAPK immuno staining to resolve the spatial relationship between MAPK vegetal blastomere, gene expression and the embryonic axes.

  gata_b	foxa
  six3/6	evx
  nk2.1

  I started to de-glycerol embryos in hourly washes of PTw around 1200.

  20/08/2015

  • 3x 10min PTx
  • 2x 45 PBT
  • 2x 30min PTx+NGS
  • 1:200 anti-mapk antibody added at 14:00

  21/08/2015

  • Wash primary antibody (afternoon)
  • 3x 5min.
  • 4x 30min.
  • Add secondary antibody anti-mouse pod at 1:250 dilution at 1800.

  22/08/2015

  • 3x 5min PBT washes.
  • 4x 30min PBT washes.
  • Overnight at 4°C in PBT.

  23/08/2015

  • Washed 3x 5min in TNT buffer.
  • Developed in 50 µL TSA + Cy5 fluorochrome for 3 min.
  • Washed 10min in detergent solution at 67°C and another with warm detergent but at room temp to cool down.
  • Washed 2x 5min PBS.
  • TDE series ending with 97% TDE in PBS with DAPI 1:1000.

  24/08/2015

  Mounted slides and checked under scope. There is quite some background with the orange filter for cy3. I cannot distinguish any signal at the posterior vegetal blastomere. Confocal showed that there was no signal from the MAPK antibody. Maybe the longer time in glycerol messed the epitopes? Or the time developing (3min) was not enough…

  DAPI (cyan), MAPK (magenta), in situ (yellow):

  

  gata b

  

  gata b

  

  gata b

  
  

  

  gata b

  

  nk2.1

  

  nk2.1

  
  

  

  nk2.1