Membranipora wnt in situ

First new try with bryozoan in situ. Separate wells for early (until extended gastrula) and late stages (early cyphonautes and later cyphonautes).

early stages 1x nk2.1 1x wnt1 1x wnt5 1x wnt7 1x wnt8
late stages 0.1x nk2.1 0.1x wnt1 0.1x wnt5 0.1x wnt7 0.1x wnt8
early stages 1x dlx 1x gbx 1x pax258 1x nanos 1x vasa
late stages 0.1x dlx 0.1x gbx 0.1x pax258 0.1x nanos 0.1x vasa

After protk step (10 min) embryos were suspended in glycine (added 800 µL) and did not sink. I added 600 µL of glycine for the second wash. The whole glycine steps lasted for 15 min. The remaining washes were a bit longer than 5 min, but I could control it better.

I noticed that the embryonic membrane dissolves with protk.

Let overnight in pre hybe.

31/03/2015

Diluted and added probes to in situ (first 0.1x then 1x). Left oven set to 63 °C (62 in the electronic thermometer and 63 in the mercury thermometer).

02/04/2015

First hybe wash I removed 1x probes first, filled with warm hybe wash and then removed 0.1x probes and added warm hybe.

  • 1x 10min hybe wash
  • 1x 50min hybe wash
  • 1x 30min 75% hybe
  • 1x 30min 50% hybe
  • 1x 30min 25% hybe
  • 1x 30min 2x SSC
  • 3x20min 0.2x SSC
  • 1x 10min 75% 0.2x SSC
  • 1x 10min 50% 0.2x SSC
  • 1x 10min 25% 0.2x SSC
  • 1x 10min PTw.
  • 4×10-15min washes in PBT (total 45min)
  • 1h in 1x blocking buffer
  • Overnight at 4°C with anti-dig-ap

03/04/2015

  • 3x 5min PBT
  • 4x 15min PBT
  • 5x 30min PTw

Developed in situ. A significant amount of pinkish background came up in my probes. wnt5 was fast, also dlx. Others had different levels of background in addition to the real signal, except Chema’s probe for NK2.1. Thus, this background is likely due to probe design or probe synthesis. Here are the lengths of the clones used to synthesize the probes:

gene nk2.1 wnt1 wnt5 wnt7 wnt8 dlx gbx pax2/5/8 nanos vasa
bp 1113 1038 1073 869 886 823 888 902 868 812

Length of the probe can be the issue but it does not quite explain the rapid background coming up. Longer probes should result in a better signal to noise ratio because the signal comes up faster and not because the background comes up slower. It can also be something related to the probe synthesis process. But what? I usually get high yields and good peak ratios. Check the numbers to see!

Ah, one obvious thing is the probe concentration. NK2.1 probe is probably more diluted than the others since it is its second or third use. Thus, the next in situ should be lower concentration or higher temperature.

04/04/15

Continued developing wnt7 and gbx and stopped them after 3h. Ethanol washes with 20min in 100% and let in PTw overnight at 4° C because glycerol was not ready.

05/04/15

Putting them in 70% glycerol with DAPI. Will put apart some of the NK2.1 embryos to do a MAPK immuno staining.