1.  Recover mouse embryos (E7.5) into 1 ml PBS

2.  Remove PBS and fix them in 1 ml 4% PFA solution at 4℃ for 30 min. 

    *temperature should be modified depending on antigen. For some antigen, room temperature or warmer temp might be better. 

3.  Wash twice with 1 ml PBT.

4.  Permealize the embryos with 1 ml ice-cold methanol (10 min, -20°C) or with 1 ml 0.2% Trion X-100/PBS (10 min on ice)

    *Fixation and permeabilization condition should be modified depending on antibody/antigen. 

5.  Wash twice with 1 ml PBT. 

    *Fixed embryos could be very sticky. The use of PBT is recommended. 

6.  Block with 500 ul blocking solution at 4℃ for 1hr.

7.  Add 100 ul 1st antibody in blocking solution with optimized concentration. 

    *volume should be modified depending on the number of embryos. 

8.  Keep at 4℃ overnight with rocking gently (or 1hr at room temperature).

9.  Wash 6 times with 1 ml PBT. 1hr for each (or can perform o/n washing) with rocking gently at 4℃.

10.  During the wash, change the tube to reduce contamination. Wash 1 time with 500 ul blocking solution.

11.  Add 100 ul 2nd antibody in blocking solution with optimized concentration.

12.  Keep at 4℃ overnight with rocking gently (or 1hr at room temperature).

13.  Wash 1 time with 1 ml PBT for 1hr at 4℃.

14.  During the wash, change the tube to reduce contamination. Wash 1 time with 500 ul blocking solution.

15.  Wash 5 times with 1 ml PBT. 1hr for each (or can perform o/n washing) with rocking gently at 4℃.

16.  Add PBT and keep it until use at 4℃.

17.  Observe the embryonic node with fluorescence microscopy by mounting the excised node onto a glass slide.

    *For excision, we use a hand-made knife made of a needle sharpened by a grinder. For mounting, we use a silicone rubber spacer. See Okada and Hirokawa, Methods Cell Biol., 2009 for detail. 

PFA solution

 4% Paraformaldehyde in PBS

PBT

 0.1% Triton X-100 in PBS

Blocking solution *Can be changed depending on the antibody. We generally use below.

TNB Buffer (0.1M Tris-HCl (pH=7.5), 0.15M NaCl, 0.5% TSA Blocking reagent (PerkinElmer, FP1020))

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