PhD notebook

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  Bruno Vellutini 18:48 on 2014/11/22 Permalink
  Tags: fluorescent, hedgehog ( 10 ), in situ ( 43 ), lmx ( 3 ), lunatic fringe ( 4 ), nk ( 16 ), pax ( 16 ), runx ( 7 ), Terebratalia transversa ( 79 ), wnt ( 60 )   

  Terebratalia segmentation in situ 

  In order to have more temporal resolution of engrailed I am adding one well per stage. In addition some segmentation related genes.

  en cleavage+blastula	en radial gastrula	en asym gastrula	en bilateral gastrula	en trilobed+early+late larva
  pax6	nk1a	nk1b	lmx1
  lfng	runx	hh

   25/11/14

  Washed probe out. Added antiDIG-AP antibody at 1700 and incubated overnight at 4 °C.

  26/11/14

  Washed antibody and started developing. Pax6 came up quite fast. I exchanged the solution after 1h and stopped after 2h. The remaining are slowly coming up, except asym and bilateral of engrailed. I used one tube of 0.8 ng/µL of probe and it must have been even lower concentration because the other 3 engrailed wells are coming up ok (same probe batch, but from another tube). Another issue is that there is not early gastrula like I wanted, they seem to be already have the differentiated lateral patches.

  Lunatic fringe and runx are not showing up. I exchanged AP from all wells except engrailed after 3h and then all wells before leaving at 4 °C overnight.

  27/11/14

  Same as yesterday with NK1s and lmx coming up nicely. Exchanged AP at 10:30.

  28/11/14

  Kept developing for 7h and stopped all wells. Nothing came up for lfng, runx or hedgehog.

  30/11/14

  Ethanol washes for all. Added 70% glycerol with 1:10000 sytox green in hedgehog well. I want to see if embryos turn red… If not I’ll check if it is better than DAPI for these regular in situs.

   
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  Bruno Vellutini 19:06 on 2014/04/11 Permalink
  Tags: fluorescent, Membranipora membranacea ( 140 )   

  Membranipora auto-fluorescence 

  So, Membranipora embryos have auto-fluorescence in the UV, green, and also a bit in the red; but not in the far red. I also checked to see if TDE gives any extra fluorescence in the samples, but no. I tested unstained embryos mounted in TDE and PBS and the fluorescence looks the same.

  

  Unstained Membranipora embryo UV filter

  

  Unstained Membranipora embryo green filter

  

  Unstained Membranipora embryo red filter

  

  Unstained Membranipora embryo far red filter

  
  
   
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  Bruno Vellutini 13:20 on 2013/10/21 Permalink
  Tags: double ( 2 ), engrailed ( 39 ), fluorescent, in situ ( 43 ), pax ( 16 ), Terebratalia transversa ( 79 ), wnt ( 60 )   

  Terebratalia additional double in situs 

  Started Terebratalia double in situs with the following selected probes. Everything looks ok.

  wnt1 (DNP) + wnt4 (DIG)	en (DNP) + wnt7 (DIG)
  wnt1 (DNP) + wnt8 (DIG)	en (DNP) + pax258 (DIG)

  22/10/13

  • Added probes to Terebratalia doubles.

  24/10/13

  • Terebratalia doubles washing day.
  • Add Terebratalia first antibody anti-dig-pod 1:250.

  25/10/13

  • Terebratalia doubles wash off first antibody.

  27/10/13

  • Terebratalia doubles develop first probe and inactivate.
  • Terebratalia doubles incubate with second antibody.

  28/10/13

  • Terebratalia doubles wash off second antibody.

  29/10/13

  • Terebratalia doubles develop second probe.
  • Terebratalia doubles stain with phallacidin.
  • Terebratalia doubles embed in TDE+DAPI.

  30/10/13

  • Mount Terebratalia doubles.

  31/10/13

  • Confocal slot for Terebratalia doubles.

  01/11/13

  Here are some scans.

  

  wnt1+wnt8

  

  wnt1+wnt8

  

  wnt1+wnt8

  

  wnt1+wnt4

  
  

  

  wnt1+wnt4

  

  en+wnt7

  

  en+wnt7

  

  en+wnt7

  
  

  

  en+wnt7

  

  en+pax258

  

  en+pax258

  
  
   
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  Bruno Vellutini 17:33 on 2013/09/13 Permalink
  Tags: aldh2 ( 3 ), fgfr ( 2 ), fluorescent, frizzled ( 7 ), in situ ( 43 ), lunatic fringe ( 4 ), pax ( 16 ), segmentation ( 10 ), Terebratalia transversa ( 79 ), wnt ( 60 )   

  Terebratalia in situ for FGFR, Frizzleds and others 

  Starting an in situ for additional Terebratalia genes related to the segmentation project. Also a double with wnt1+wnt5.

  FGFR	Pax2/5/8	Frizzled2	Frizzled4
  Frizzled10	LFNG	ALDH2	Wnt1+Wnt5

  14/09/13

  Put probe.

  16/09/13

  Washed in situ and added anti-dig-ap 1:5000 to the first seven genes and anti-dig-pod 1:250 for the double fluorescent.

  17/09/13

  • Washed antibody.
  • Set Cy3 TSA to double fluorescent for 1h45.
  • Started developing regular NBT/BCIP at 16h.
  • Stopped Fz2 around 20h, let the rest overnight.

  18/09/13

  • Stopped FGFR and Fz4. Let the rest continue, almost stopped Pax2/5/8, since it is almost there.

  22/09/13

  • Ethanol washes.
  • Glycerol added.
   
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  Bruno Vellutini 19:15 on 2013/03/08 Permalink
  Tags: engrailed ( 39 ), fluorescent, in situ ( 43 ), Terebratalia transversa ( 79 ), wnt ( 60 )   

  Double in situ in Terebratalia 

  Starting double in situ with probes correctly diluted to 1 ng/µL. Stages sampled are: late blastula, early/mid/late gastrula, trilobed, early/late larva.

  en-DNP+wnt1-DIG-POD

  en-DNP+wnt5-DIG-POD

  Regular beginning. I left 5 minutes of the ProtK shaking, mistakenly.

  11/03/13

  Regular washing. Put Anti-DIG-POD diluted 1:250 (in 250 µL each well).

  12/03/13

  Began washing at 11h30. Did extra wash with PBT and also a couple of extras for PTw.

  13/03/13

  Developed first probe with Cy3 and stopped the reaction with 1 detergent wash of 5 min and another detergent wash of 10 min. Specific expression can be seen in the stereoscope. Put the Anti-DNP antibody for the second probe.

  14/03/13

  Just washed thoroughly.

  15/03/13

  Develop the second.

  WORKS! engrailed=green, wnt1=red

  

   
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  Bruno Vellutini 19:27 on 2013/03/07 Permalink
  Tags: confocal ( 23 ), engrailed ( 39 ), fluorescent, in situ ( 43 ), Terebratalia transversa ( 79 )   

  Fluorescent in situ of Terebratalia engrailed 

  Mounted a slide with Terebratalia embryos from the first fluorescent/double in situ I tried for the segmentation genes and observed under the confocal. Mounting media had DAPI (magenta) and en is green. Although there is background it is possible to distinguish the real signal.

  Here are some max-projections to illustrate:

  

   
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  Bruno Vellutini 17:20 on 2013/02/22 Permalink
  Tags: distal-less ( 11 ), double ( 2 ), engrailed ( 39 ), fluorescent, in situ ( 43 ), Terebratalia transversa ( 79 ), wnt ( 60 )   

  Terebratalia fluorescent and double in situs for segmentation 

  Starting a new fluorescent and double in situ for segmentation genes. Standard procedures.

  wnt1	wnt5	dlx	en (DNP)
  en + wnt1	en + wnt5	en + dlx

  23/02/13

  Probes put at 14h.

  25/02/13

  Washing day. I put the DIG-POD for all wells except en with DNP-POD (HRP).

  26/02/13

  Regular washes.

  27/02/13

  Developed first probe in Cy3 for 1h45. Stopped the reaction 2x 5 min in detergent solution at 62 °C. After washes I checked samples in the stereoscope, but could not see any clear signal in any of the samples. Doubles were blocked and incubated with second antibody (DNP-POD).

  28/02/13

  Developed second probes from doubles in Cy5. Samples were stopped in the same manner and further washed in PTw. I stained with DAPI 1:1000 in PBS for 20 min, washed 3x in PBT and stored in PTw. I mounted 1 slide with en+wnt5 and had a look under the scope.

  It seems that en with DNP-HRP has no background, but the signal was really faint. This might be do re-using the 500 µL of a 0.5 ng/µL probe (find out later that it was half diluted…).

  

  The wnt5 with DIG-POD resulted in a dirty and shiny larva with background all around. Cannot see any real signal. So it seems that DIG-POD needs to be washed longer or there is something wrong with the antibody or TSA kit.

  

  DAPI staining was ok…