Immunofluorescence staining

Materials: Use freshly prepared solution for each batch of slides
 

Paraffin Embedded Organoid Sections on glass slides
Note: It is better to embed the Matrigel containing organoids in Histogel first (see manufacturer’s instructions), then process for paraffin embedding and sectioning
 

Wash Buffer = 0.1% Tween in TBS
      
Unmasking Solution = Citrate Buffer (pH 6.0)
     
Power block = 5% normal serum in TBS (dependent on which animal the secondary antibody was raised; if raised in goat, then goat normal serum)
 

De-paraffinize (start around 2 pm)
1. Xylene 1 → 20 minutes 
2. Xylene 2 → 20 minutes
3. 100% EtOH → 5 minutes
4.  90% EtOH → 5 minutes
5.  70% EtOH → 5 minutes
6.  50% EtOH → 5 minutes
7.  30% EtOH → 5 minutes
8.  Distilled water → 5 minutes x2
 

Antigen Retrieval 
9. Add slides to pre-heated Unmasking Solution between 95°C - 100°C for 20 min in a heated water bath
10. Slowly let the unmasking solution with the slides cool to room temperature, 20-30 min
11. Rinse in diH20 gently x1
 

Blocking
12. Rinse with wash buffer x3 changes
13. Circle sections with pap pen
14. Power block for 30-60 min at room temperature
15. Gently wipe away excess Power block; do no wash
     Note: Do not let the sections dry at any point until mounting with coverslips
 

Antibodies
16. Add primary antibody/isotype control (diluted in TBS with 1% normal serum) overnight at 4°C in humidified chamber.

TITRATE Primary Antibody before use (or used optimized dilution):
1:50 = 8 µL Ab + 392 µL TBS
1:100 = 200 µL Ab @ 1:50 + 200 µL TBS 
1:200 = 200 µL Ab @ 1:100 + 200 µL TBS
1:400 = 200 µL Ab @ 1:200 + 200 µL TBS
1:800 = 200 µL Ab @ 1:400 + 200 µL TBS 
etc.
0 = 200 µL TBS and/or TBS containing primary antibody isotype control with similar dilutions/concentrations
 

17. Place slides in wash buffer x3 for 10 min each 
18. Add fluorescently labeled (Alexa Fluor 488 or 594 or else) secondary antibody (2-10 μg/ml) on the sections and incubate for 1-2 hr at room temperature in dark.

     Note:  If immunostaining for a second antigen/epitope on the same section, do it in a sequential manner. Do not mix any two primary or secondary antibodies. Finish staining with first primary antibody and first secondary antibody, wash buffer x3, then again apply respective Power block and finish staining with second primary antibody and second secondary antibody. Perform nuclear counterstain at the end.

19. Place slides in wash buffer x3 for 10 min each 
20. Gently rinse in TBS x1
21. Add Hoechst 33342 (nuclear counterstain; 1: 1000 – 1:2000 in TBS) for 5-10 min in the dark at room temperature.
22. Gently rinse in TBS x3
23. Mount sections in Vectashield. Seal coverslip edges with clear nail polish. 
24. Store slides at 4°C