Immunofluorescence staining

We used this protocol for most of our IF staining. The concentration of the primary antibody is in the range of 1:100 to 1:200.
 

EdU-GFP-WGA-DAPI Protocol

EdU incorporation

1. Dissove EdU with DMSO, add saline to make the 10mg/ml EdU working solution. (5% DMSO)
2. Pig was injected with 100ml EdU solution by ear vein (total 1g per injection) at day 1, day 10 and day 20 before sacrifice.
3. Harvest the heart tissue and fixed in 10% formalin and processed for paraffin embedding.
    

Dehydration and Antigen Retrieval

1. Xylene x 2, 10 min
2. 100% EtOH x 2, 5 min
3. 95% EtOH x 2, 3 min
4. 95% EtOH, 3% H2O2, 15 min
5. 70% EtOH, 2 min
6. Wash slides in PBS, 5 min
7. Place slides in coplin jar with antigen retrieval solution and heat in pressure cooker at maximum pressure for 10 minutes.
8. Release the pressure and carefully remove coplin jar from pressure cooker.  Allow slides to reach room temperature before continuing staining.  –usually 20min
9. Wash in 1X PBS
10. Using a liquid blocking pen, circle each section on the slides.
11. Place slide in a humid incubation chamber and add 1x PBS to each section.  *NEVER LET SECTIONS DRY DURING THE STAIN PROCESS*
12. Wash with 0.1% PBST, 3 times 2min each
13. Wash with 0.5% PBST, 1 time 10min 
     

EdU Staining

    1. Add Edu staining mix, approx 200ul per slide; make just before usage

        500 ulEdU staining mix:  1X Buffer              430 ul

                                                CuSo4                  20 ul

                                                Alexa 643 Azide   1.2 ul  (light sensitive)

                                                 1X EdU Additive   50 ul  (10X stored in -20degreeC, make 1X with water     fresh each time)

    2. Incubate for 30 minutes at room temperature, keep DARK.

GFP and DAPI 

1. Wash slides three times with 1x PBS, 2 minutes per wash
2. Add blocking solution (10% donkey serum in PBST) to each slide.
3. Incubate for 10 minutes at room temperature.
4. Remove blocking solution and add chicken-GFP antibody ; 1:200 in blocking solution
5. Incubate for 30 min at room temperature
6. Wash slides twice with 1x PBS, 2 minutes per wash
7. Add biotinylated anti-chicken secondary with fluorescence (1:200) in blocking buffer
8. Incubate for 40 minutes at room temperature
9. Wash slides twice with 1x PBS, 2 minutes per wash
10. Add DAPI (1:500; 2ug/ml final concentration), streptavidin-488 (1:200) and WGA Rhodamin (547) (1:200) in 1x PBS
11. Incubate for 10 minutes at room temperature.
12. Wash slides twice with 1x PBS, 2 minutes per wash
13. Mount-DAKO mounting medium
     

Product List:

-EdU  SantaCruz (sc-284628A)

-EdU staining kit Invitrogen (C10340)

- DAKO mounting medium S3023

-Donkey serum Sigma D9663

-WGA-Rhodamin Vector Lab (RL-1022)

-Antigen-retrieval buffer Vector lab H-3300-250

Imaging

We usually use four channels Blue (DAPI) 405; Green (GFP) 488; Red (WGA) 546; Far red (EdU) 647.