Immunofluorescence

Immunofluorescence

Materials and Reagents

1. primary antibody and FITC-conjugated secondary antibody (Life Technologies, Invitrogen)
2. 4% PFA
3. filtered phosphate buffered saline (PBS)
4. cytoskeleton (CSK) buffer: 50 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, 0.5% Triton X-100, and 10 mM Pipes (pH 6.8)
5. Blocking buffer: 10% goat serum, 1% BSA in PBS.
6. 30% sucrose in PBS
7. DAPI 
8. Click-iT EdU Kit (Roche)

Procedure

1. Pregnant mice were intraperitoneally injected with EdU at 100 μg/g body weight.
2.  Embryo is isolated from the pregnant mice in PBS. And than, the brain isolated from embryo is fixed by 4% PFA at 4°C.
3.  After 24 hr fixation, perfuse the tissue with 30% Sucrose Solution overnight at 4°C.
4. For dissecting the tissue, mount in OCT embedding compound at -20 to -80 °C and cut 10 µm thick tissue sections using a cryostat.
5. To dry the slides for 30 minutes on a slide warmer at 45 °C. Frozen sections  can be stored at -80 °C.
6. At the beginning of staining, wash slides twice with PBS for 10 minutes to remove the OCT.
7. To permeabilize the tissue, perfuse the sections with CSK buffer for 30 minutes.
8. To block non-specific binding, incubate the tissue sections with blocking buffer for 30 minutes at room temperature.
9. Add primary antibody diluted in blocking buffer to the sections and incubate overnight at 4°C in humidified chamber.
10. Wash sections twice with 1X PBS (with or without 0.1 % Triton X-100)  for 10 minutes each.
11. Add secondary antibodies and DAPI (1 μg/ml) diluted in blocking buffer to the sections and incubate at room temperature for one hour.
12. Wash sections three times with 1X PBS (with or without 0.1 % Triton X-100)  for 10 minutes each.
13. Following the  Click-iT EdU Kit (Roche) protocol to detect the incorporated EdU.
14. Wash sections three times with 1X PBS (with or without 0.1 % Triton X-100)  for 10 minutes each.
15. Add mounting medium to the slide and place a coverslip on the tissue sections. Next, circle the edges of the coverslip with clear fingernail polish to prevent the cells from drying. 
16. To examine the slides under a microscope.