In-place hybridization (in the case of zebrafish)

fixed
collect zebrafish embryos, culture

in Holfretor water, remove the egg membrane with
protease
when the desired development period is reached, secure with 4% polyformaldehyde, in Stored at 4 degrees C, washed in 24 hours with 50% methanol 2% polyformaldehyde solution, then replaced with methanol, stored at -20C, to be used (two days and more than two days of embryos need to be treated with hydrogen peroxide, remove pigment. Or culturephenyls and pyrides, which can block the formation of )
. In-place hybridization
First day
1. Rehydration and fixation
1) Absorb the fixed embryo, add 50% methanol to the PBST solution, and place for 5 minutes.
2) Replace with 30% methanol PST solution, place 5 minutes
3) replace with PBST solution, place for 5 minutes, repeat
4) replace with 4% polyformaldehyde PBS solution fixed for 20 minutes
5) wash twice with PBST, each time for 5 minutes, room temperature.protease treatment with post-fixation (this experiment does not do this step)
1) using 10ul/ml protease K to process the embryo at room temperature. Embryos below 5 bodies are not processed, embryos under 5 bodies are processed for 3 minutes from 5 bodies to 24 hours, and embryos processed for more than 24 hours are processed for 5 minutes or more. The shorter the development time of the embryo, can not use or less protease treatment, the longer the development of the embryo needs to use protease to loosen
tissue
, in order to facilitate hybridization.
2) Wash lightly with the PBST solution and place in the PBST for 5 minutes.
3) Secure with a PBS solution of 4% polyformaldehyde for 20 minutes, and wash twice at room temperature
4) at PBST for five minutes at room temperature.
2. Pre-hybridization
1) Each tube is replaced with approximately 300ulHYB-solution, 60C water bath for 5 minutes to avoid oscillations.
2) replaces HYB-with an equal volume of HYB-.
3) 60 degrees C water bath, pre-hybridization for more than 4 hours..3. hybrid
1) sucks away the pre-hybrid HYB plus 100ul added probe to the HYB plus solution (probe concentration is about 1 ng/ul).
2) 60C temperature bath overnight.
: the temperature of hybridization and pre-hybridization can be 55 to 60 degrees, the lower the temperature, the better the probe binding, the higher the temperature, the smaller the background.
II. In-place hybridization is the second day
.1.
1) Recycle the probe and store it at -20C (usually the probe can be reused about ten times).
2) Add 50% methamide/2XSSCT solution 1 ml, 60 oC, place for 30 minutes, repeat.
3) Replace 2XSSCT1ml, 60C, for 15 minutes.
4) replace 0.2XSSCT1ml, 60 degrees C, place for 30 minutes, repeat..2.
1) Wash twice with MABT, for five minutes at a time,
the
shaker and shake gently.
2) add 1 ml 1:2:7 solution at room temperature for one hour.
3) Add enzymatic high-acid antibodies to a 1:2:7 solution at a ratio of 1:3000 , 4C refrigerator overnight.
3. In-place hybridization day 3
1) replace the antibody solution with 1 ml of MABT solution containing 10% thermal inactivation
serum
, place it on a shaker for 25 minutes, then replace it with 1 ml MABT, 25 minutes, replace it with 1 ml MABT solution, for more than one hour, and finally replace it with 1 ml MABT solution for 25 minutes.
2) Wash the embryo three times with a 1 ml 1mM L-meter staining buffer for five minutes
3) to suck up the staining buffer, plus 300ul BM Purple AP Substrate (bottom, with 5mM L-mitole), and a tin foil on the outer bread of the sixteen-hole plate to avoid light, avoid shaking, and reduce room temperature.
4) Every hour to observe whether the embryo began to color
5) will be fully colored embryo substrate sucked out, washed two or three times with PBST plus 4% polyformaldehyde fixed, take pictures.
6) 4C refrigerator.solution in in-place hybridization:
PBS:
NaCl 8g
KCl 0.2g
Na2HPO4 1.44g
KH2PO4 0.24g
DEPC H2O 1L
HCl PH to 7.4
filtration, sterilization 4% polyformaldehyde:
polyformaldehyde 40g
PBS 1L heating
continuously stirred until the solution is clarified. -20C SavePST:
PBS solution plus Tween-20 to give its final concentration 0.1%.20XSSC:
Na3Citrate 2H2O 88.2g
NaCl 175.5g
DEPC H2O to 1L
filtration, sterilization SSCT:
SSC plus Tween-20 for a final concentration of 0.1%..HYB-:
: 20XSSC reservoir: DEPC water : 2:1:1 preparation
add Tween-20 to make its final concentration 0.1%, -20c save..HYB:
HYB- 20ml
yeast RNA 10mg
heparin 1mg.
at -20 degrees C. . MAB:
maleic acid 11.6g
NaCl 8.8g
with solid NaOH (about 7g) tuned to Ph=7.5
4C save MABT
MAB plus Tween-20 to make its final concentration 0.1%. 10% blocking reagent:
blocking reagent 8g
MAB 72ml 1:2:7 Solution:
Inactivate sheep serum: 10% BM blocking reagent: MABT s1:2:7
Timed outStaining buffer:
Tris 12.1g
pH9.5
Mgcl 6H2O 10.2g,
Nacl 5.85g
Tween-20 1ml,
use the 1M L-Mimi reservoir before using, so that the final concentration of 1mM
.