method bannerreturn to methods indexreturn to methods index

 

 

 

 

 

Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu


Distal-less antibody protocol

The Dll antibody is affinity purified from rabbit sera, and is at ~200 ug/ml.
Please store it at 4oC.
It can be reused 2-3 times if stored in PBT with NaN3.

Reagents needed:
Fix Block Wash
10x PBS, pH 7.0
(200mM KPO4, 140 mM NaCl)
500 mM EGTA pH 7.4
37% formaldehyde (Sigma)
10x PBS
Triton X-100
BSA
DMSO
10x PBS
Triton X-100
Developer
Metal-enhanced DAB solutions (Pierce)

Other
methanol
30% H2O2 (Sigma)
goat anti-rabbit peroxidase (Jackson) OR
goat anti rabbit biotin and Elite ABC reagents (Vector)

Methods
1) Make a stock soln which is 1.33x PBS and 67 mM EGTA ("fix buffer"). This can be filtered sterilized and stored for ~ 1 yr.

2) Mix 3 parts fix buffer with 1 part 37% formaldehyde just prior to use. Fix PBS washed, dissected embryos for 20 min-8 hrs (20 min for small arthropod embryos; 6-8 hrs for larger vertebrate embryos).

3) Wash embryos 3-4x with MeOH to remove fix. Treat for 1(arthropods) 5(vertebrates) min with 3% H2O2 in MeOH to inactivate endogenous peroxidases. Wash away H2O2 with 4-5 changes of MeOH. Fixed embryos can be stored in MeOH at -20oC indefinitely.

4) Rehydrate embryos in 50% 1x PBS pH 7, 50% MeOH, then 100% 1x PBS.

5) Block embryos in PBT (1x PBS pH 7.0, 0.1% Triton X-100, 2% BSA) for 1 2 hrs at 4oC. PBT spoils very quickly; I make up 50 mls at a time (see step 7) and store it at 4oC for 2-3 days For vertebrates, 5% DMSO can be added to the blocking soln to permeabilize the embryos. Some arthropod embryos need to be sonicated to permeabilize them. This should be done while they are in block. The best staining is achieved, if embryos are sonicated in brief (1-5 sec) pulses until ~50% are visibly disintegrating.

6) Incubate embryos overnight at 4oC with 1 (arthropods) or 5 (vertebrates) ug/ml anti-Dll AB in PBT (no DMSO). I typically stain ~ 30 ul of packed arthropod embryos in 600 ul of AB or 10 vertebrate embryos in 5 mls of AB. The incubation can be done with no, or gentle, shaking. (Stock AB soln is 200 ug/ml)

7) Wash embryos 10x over 1 (arthropods) or 2 (vertebrates) hrs with PT (1x PBS pH 7.0, 0.1% Triton X-100; I make this soln up in 1 liter volumes, filter sterilize it, and store it at room temp. To make PBT, I put 50 mls of PT in a 50 ml conical and add 1 g of BSA).

8) Incubate embryos with 1:400 goat anti-rabbit peroxidase (or goat anti rabbit biotin) for 1 (arthropods) or 4 (vertebrates) hrs at 4oC.

9) Wash as before.

10) Develop (or add premixed Elite A and B reagents at 1:500 each for 30 min, then wash again, then develop) in Pierce DAB solns according to the manufacturer's instructions (i.e. mix 9 parts of their buffer with 1 part of their metal-enhanced DAB, and add it to the embryos). This step goes very rapidly (i.e. in 1-2 min). Be prepared to wash away the developer with PT. You can slow the reaction down by using less of the DAB soln or by carrying out the developing step on ice.

11) Wash embryos 5x with PT, clear, store and mount in 10-50% glycerol, 100 mM Tris pH 8, 0.02% NaN3.