In situ for Membranipora NANOS, PIWIL2, TDRD1, PUM, MAEL


Warning: preg_match_all(): Compilation failed: invalid range in character class at offset 7 in /home/customer/www/phd.organelas.com/public_html/wp-content/themes/p2/inc/mentions.php on line 77

Samples

  • Mm Early: 2-128 cells + day 1 + day 3-4 + late cleavage
  • Mm Late: pre hatch + hatched + day 5 + day 7 + early and late cyphonautes

26/02/2012 — Day 1

Samples were put in 2 eppendorfs (early/late) and rehydration, PTw washes and first prehybe carried on there. So between washes there was a gap to wait the embryos to settle.

On the last step, addition of hybe buffer, samples were aliquot to the following 24-well plate:

NANOS PIWIL2 MAEL PUM TDRD1
EARLY
LATE

Left overnight at 62 °C at 18h.

27/02/2012 — Day 2

Probes were already diluted at the stock concentration 50 ng/µL and I had to dilute MAEL, PUM, and TDRD1 to 1 ng/µL. Hybridization started at 11:30.

29/02/2012 — Day 4

Washes with a few modifications (due to busy day):

  • 60 min instead of 30 min at 25% hybe 75% 2X SSC (during Sars Seminar).
  • 3x 10 min 2X SSC at hybe temp.
  • PTw washes reduced to 5 min.
  • PBT washes reduced to 5 min.

Anti-DIG put at 17:10, rocker at 4 °C.

01/03/2012 — Day 5

Regular washes.

02/03/2012 — Day 6

Developing started at 12:30 (~450 µL AP substrate solution).

16:00 — Some signal seems to be appearing in MAEL. Early stages not so conclusive, but on late stages look like a bilateral pattern is emerging in the lateral flanks of the cyphonautes.

03/03/2012

16:00 — All five wells with late stages showing cyphonautes with the same bilateral pattern observed above.

04/03/2012

Whole day at 4 °C.