adam /28.07.2005 10:45/

Зачем нужна прегибридизация????? Подскажите

LMP /28.07.2005 20:49/

иногда рассматривается как "забивка"

Гость /01.02.2007 20:25/

In situ hybridization protocol for Drosophila embryos.

For digoxygenin labeled RNA probes
Incorporating maleic acid blocking buffer


A. Collection, preparation and fixation of embryos

1) Collect and rinse embryos in H2O or Drosowash (0.7%NaCl, 0.03% Triton X100)

2) Dechorionate for 3 min in 50% Clorox. Wash thoroughly. (I wash for 1- 1.5 min under running distillate water, then dry on paper towel).


3) Fix embryos for 50 min in

1.35 ml 37% formaldehyde
0.5 ml 10X PBS
0.5 ml 0.5 M EGTA
2.65 ml H2O
5 ml heptane
Make this solution fresh every time. Rotate or shake embryos constantly.

4) Devitellinize the embryos by removing the aqueous phase (bottom) and adding about 15 ml of methanol. Shake vigorously for 30 sec to several min. Devitellinized embryos will sink.
- Rinse embryos with 5 ml of methanol, then 2 times with 5 ml of ethanol.
Store at –20oC. Longer storage time may decrease background. I have used embryos that were stored for 1 year without noticeable loss of signal.


B. Pre treatment of embryos.
Make 100 ml HMHyb (look at buffer page). Aliquot into 15 ml. Store at –20oC
Make 1ml 100 mg/ml glycine (100 mg glycine and 1 ml H2O)

Make fresh solutions. For 5 samples:

1X PBT 1:1 HMHyb/PBT
10 ml 10X PBS 5 ml HMHyb
500 ml 20% tween 20 5 ml PBT
90 ml H2O
filter sterilize

10ml/ml proteinase K in PBT PBT + 2 mg/ml glycine
5ml ~15 mg/ml proteinase K, “Roche” (at 4oC) 200ml 100mg/ml glycine
5 ml PBT 10 ml PBT

50 % xylene / 50 % ethanol
5 ml xylene
5 ml ethanol

90% xylene/ 10% ethanol
9 ml xylene
1 ml ethanol

PBT-5% formaldehyde
20 ml 1X PBT
2.631 ml 37% formaldehyde

3:1 MeOH/ PBT-5% formaldehyde
6 ml MeOH
2 ml PBT-5% formaldehyde

2:2 MeOH/ PBT-5% formaldehyde
4 ml MeOH
4 ml PBT-5% formaldehyde

1:3 MeOH/ PBT-5% formaldehyde
2 ml MeOH
6 ml PBT-5% formaldehyde

1) warm up embryos to room temperature.

2) Rinse in 50% EtOH/50% xylene, then soak in 90% xylene/10% ethanol for several hours (at least 1 hour). Rinse again in 50% EtOH/50% xylene then several times in ethanol. This step is supposed to reduce background.

3) Rinse twice in MeOH, and then 2 min in 3:1 MeOH/ PBT-5% formaldehyde; 2 min in 2:2 MeOH/ PBT-5% formaldehyde; 2 min in1:3 MeOH/ PBT-5% formaldehyde. Post-fix for 30 min in PBT-5% formaldehyde. (The series is supposed to help with early embryos).

4) Rinse 3 times in PBT (2 min each).

5) Incubate 3 min with 10mg/ml proteinase K in PBT. Thins step is essential and lowers background but too long of incubation will cause the embryos to break up in hybridizations.

6) Stop by quick rinsing in PBT + 2 mg/ml glycine, and then washing embryos 1 time for 5 min with PBT + 2 mg/ml glycine.

7) Rinse 1 time with PBT, then wash for 2 min with PBT.

8) Post-fix 30 min in PBT + 5% formaldehyde.

9) Rinse 1 time with PBT, then wash 4 times for 2 min with PBT.

C. Pre Hybridization, hybridization, and washing.

1) Rinse in 50% HMHyb/50% PBT, then in HMHyb buffer. Place embryos at 55oC for 3 hours. (Check the temperature at thermostat).

2) Replace HMHyb with fresh HMHyb. Pre hybridize at 55oC for at least 1 hour. Embryos can stored in HMHyb for at least 2 weeks at -20 oC.

3) Replace HMHyb with 200ml HMHyb + 2ml probe in HMHyb. Hybridize overnight at 55oC. Mix a few times over an hour before going home and a few times when you return the next day.

(The optimum temperature varies depending on the probe. For the SxlPe probe 50oC seems to be optimum. If you use a lower temperature, do the subsequent washes at the alternative temperature. If you have a long GC rich probe you might want to raise the hyb temp to 65oC. This will not damage the embryos.)


Make 300 ml 1X MAB +0.1% tween 20 buffer, filter sterilize into several tubes.

300 ml 1X MAB +0.1% tween 20 buffer
(100 mM maleic acid, 150 mM NaCl, pH=7.5)

2.629g NaCl
3.483g maleic acid (Sigma)
300 ml H2O
pH=7.5

Mix NaCl and maleic acid in 250 ml H2O and start adding solid NaOH until solution begin to clear at pH~6. Then finish titration to pH= 7.5 with liquid NaOH.
(this takes enormous amount of base to pH).

Add volume to 300 ml.
Add 1.5 ml 20% tween 20

Filter sterilize into several tubes.









Unfreeze 10% BMB (Boehringer Mannheim Blocking Reagent) at 37oC and aliquot it into several tubes, ~ 5 ml in each.

MAB +2% BMB
10 ml 1X MAB
2 ml BMB

4) Wash embryos 4 times for 30 min with warm (50oC) HMHyb, then 4 times with 1X MAB (maleic acid buffer + 0.1% Tween 20 = 100 mM maleic acid, 150 mM NaCl, pH=7.5).

Detection.

1) Block for 2 hours with MAB + 2% Boehringer Mannheim Blocking Reagent

2) Block for 1.5 hours with MAB + 2% Boehringer Mannheim Blocking Reagent

3) Add pre-adsorbed alkaline phosphatase couples anti-digoxygenin antibody to a final dilution of 1:2000. Typically I add 5ml of 1:10 Ab to 1.0 ml MAB +2%BMB. Rotate overnight at 4oC.

1.5 ml of Ab
2.5 ml MAB +2%BMB
rotate for 30 min at room temperature
add 0.5 ml to each tube with embryos.

watchesonline89 /20.12.2022 16:12/