Immunofluorescence of Organoids in 8-chamber slides

MATERIALS (Source information in Key Resource Table): 

• 4% PFA                                                                                         · DAPI
• 100% Methanol                                                                             · ProLong Glass
• 1X PBS                                                                                          · Glass coverslips
• 30mM Glycine in 1X PBS                                                              · Clear nail polish
• Blocking Buffer (1X PBS, 2 mg/ml BSA, 0.1% Triton X-100)          8-chamber slide
• Primary and Secondary Antibodies

1)  Dissociate organoids into single cells or very small clusters of cells.

2)  Seed 2.5 E4 organoid-derived singles cells per well in 8 µL of Matrigel in an 8-chamber slide. Matrigel should be spread into a small circle, do not touch edge of chamber sections. Note: Seeding density may vary depending on size of cells. 

3)  Allow organoids to grow 4-6 days to desired size and fix the organoids choosing the appropriate fixation method for your target of choice (step 4 or 5 below).

4)  PFA Fixation:

• Fix Organoids with 200µL of 4% PFA in 1X PBS for 15min @ RT
• Quench with 200µL 30mM Glycine for 5min @ RT

5)  Methanol Fixation: 

• Rinse well with 200 µL of 1X PBS @ RT
• Aspirate PBS and fix using 200 µL of ice-cold 100% MeOH 
• Incubate at -20°C for 20 min. (If slide is shared with wells that require PFA fixation, perform incubation on ice.) 

6)  Wash 3X with 200µL 1X PBS for 5min each wash

7)  Add 200µL Blocking Buffer for Permeabilization for 1hr @ RT

8)  Aspirate and add 100µL of primary antibody dilution (see key resource table for dilution information) in blocking buffer and incubate at 4C overnight. Parafilm the slide. 

9)  Aspirate 1° antibody and wash 3X for 5min with 200µL 1X PBS at RT.

10) Prepare secondary antibody (see key resource table for dilution) and DAPI (1:500) in Blocking Buffer. Incubate for 2 hours at RT. (Protect from light)

11) Wash 3X with 200µL 1X PBS for 5min each wash

12) Snap off the plastic brackets that hold the chambers to the slide. Hold the slide in one hand and gently pull straight up on the chamber with the other hand. 

13) Once the slide is clear of the chamber insert, add 15 µL of ProLong Glass to every well of the 8-Chamber slide. Note: Add ProLong to base of 8-Chamber Slide along hydrophobic barrier.

14) Cover with glass coverslip.

• Place edge to base of 8-Chamber Slide to create uniform contact with ProLong
• Slowly cover 8-Chamber Slide to avoid trapping air bubbles
• Let cure overnight at room temperature in the dark

15) Seal the coverslip with clear nail polish