RMO44 Antibody
Staining Protocol, Moens Lab
1.
At 24 hrs
of development (prior to the formation of pigment), change embryo medium.
Culture embryos in 0.003 % PTU (phenyl-thio urea) dissolved in Embryo Medium to
prevent pigment formation.
2.
Dechorionate
embryos and allow to straighten (PTU makes chorions somewhat brittle). Fix 30-40 embryos/tube at 48
hr of development in 2% TCA (trichloracetic acid) made in 1X PBS. Fixation
should last 3 hr. at room temp. Embryos will turn white within 5 minutes of
initiating fixation.
3.
Wash
embryos 2 times 5 minutes with 1 ml 1X PB at room temp.
4.
Wash
embryos 3 times 5 minutes with 1 ml 1X PBTriton with 0.5% TritonX-100
(PB-Triton).
5.
Block
non-specific binding sites using 1 ml PB-Triton+ 10% goat serum + 0.1 % BSA
(Blocking Solution) for 1 hr at room temperature.
6.
Add 0.3 ml
per tube of Antibody. Either:
a)1:1 mix of unused RMO44 (in Cecilia Ab
box at 4oC) with Blocking Soln ?OR-
b) reuse aliquot of RMO44 (in Andrew Ab
box at 4oC). Check to make sure old RMO44 is NOT cloudy. Antibody
should be able to be reused 5-10 times (note tick marks on top of tube).
Incubate with RMO44 overnight at 4oC.
7.
Remove
RMO44 Antibody and SAVE, marking number of uses on top of tube.
8.
Wash
embryos 6 times 15 minutes each with PBTriton.
9.
Dilute
Secondary Ab (Goat anti mouse-HRP conjugated) 1:200 in Blocking Solution. Add
to 0.3 ml/tube embryos and incubate at room temp. 5 hrs. at room temp. OR
overnight
10.
Wash
embryos 6 times 15 minutes each with PBTriton.
11.
Wash 2
times with PBS-0.5% DMSO.
12.
Dilute
Tyramide 1/100 in Diluent. Tyramide/diluent located in NEN kit which is in 4oC
deli case.
Develop 3 minutes at room temp with approx 0.3 ml dilute
tyramide per tube.
13.
Wash 4x 10
min with PBTriton. Clear by washing 10 minutes in 30% glycerol/Tris then 50%
glycerol/Tris.
14.
Store in
dark in deli case. Deyolk and score on flurorescent microscope.
PB
= 0.1 M phosphate buffer pH 7.4
PBTriton
= PB with 0.5% Triton-X-100
