Double Immunostaining via antigen retrieval

1. Fix the tissue for 4 hours @ RT in 4% MEMFA (Made in water)
2. Wash the tissue with PBS 3x10' @ RT on a shaker
3. Put in 30% sucrose and shake O/N at 4˚C (cold room)
4. Embed in OCT and freeze on Dry ice and transfer the frozen block to -20˚C freezer until sectioning
5. Cut sections as desired, let them dry for at least 1 hour @ RT. Contour with Dako pen
6. Wash the slides 3x10' in PBS-Tween 20 (0.3% Tween20) in plastic container
7. Block for 1h @ RT in PBS, 1% BSA, 0.3% Triton X100 (blocking buffer)
8. Add RFP/Cherry/GFP/ or any other antibody (MBP 1:200, b3TUB 1:200, MHC, MEF2c etc)that does not require antigen retrieval diluted in blocking buffer and dispense 250-300µl per slide. Incubate O/N in the cold room.
9. Wash 5x10' with PBS-Tween 20 at RT in plastic container
10. Add (250-300µl per slide) secondary antibody (Alexa's) diluted in blocking buffer (1:200 dilution) and incubate for 1h @ RT
11. Mount in 50% Glycerol-­‐PBS. Put coverslips on the slides but don't put nail Polish. Image the slides if possible to be on the safe side and to make sure the staining worked.
    1. If you don't want to image then just go from step 10 to 13.
12. Wash the slides 5x10’ in PBS-­‐Tween 20 (0.3% Tween20) in plastic container. This step will allow the coverslip to slide away from the slides. Carefully take out the slides after the first wash and the coverslip will just slide away and remain in the washing container.
13. Expose slides to 25 min of Glycine solution (200mM glycine, 1XPBS with 0.3% TritonX-­‐100). This is on my shelf(1 liter bottle-­‐works fine although made last year)
14. Prewarm water bath to 86ºC. Warm up the plastic container (5 slide holder) containing citrate buffer (Citrate pH6-­‐DakoCytomation 1:10 diluted in water). The stock is in Andrea’s fridge. Dilute it 1:10 and then warm it up in plastic container from my drawers. I warmed up the water bath first thing in the morning and left the citrate buffer in there to be really warmed up before putting the slides in there.
15. Put slides in the warmed up citrate buffer container and put it back into the 86ºC and start the timer for 15 mins.
16. Take the whole plastic container out containing the slides and leave it on the bench at RT for 15 mins.
17. Block for 1h @ RT in PBS, 1%BSA, 0.3% TritonX100 (blocking buffer).
18. Add Sox2 (1:500), Col2a1 (1:20), Keratin (undiluted) antibody/ies in Blocking buffer and dispense 250-­‐300μl per slide. Incubate O/N in the cold room (4ºC).
19. Wash 5x10’ with PBS-­‐Tween 20 at RT in plastic container.
20. Add (250-­‐300 μl per slide) secondary antibody (Alexa’s) diluted in blocking buffer (1:200 dilution) and incubate for 1h at RT.
    1. You can check quickly under the DAPI channel if the nuclei are still visible, if not then just add the Hoechst again along with secondary antibody. If you did not image the slides after the first staining, then you don’t need to add Hoechst in the first staining and just add it here.
21. Wash 3x10’ with PBS-­‐Tween 20 in plastic container.
22. Mount in 50%Glycerol-­‐PBS. Put coverslips on the slides and seal with nail polish.
23. Good luck Imaging :)

I have tried this protocol combining a Cherry antibody as first staining and Col2 as second and the immunofluorescence signal from Cherry was visible and intact after the second round of staining with antigen retrieval.