Immunofluorescence for tdTomato-expressing cells

Immunofluorescence on Paraffin Sections

Using anti-mCherry Ab (also detects tdTomato)

Mariani Lab (F. Mariani and M. Serowoky)

12/10/2021

Reagents and solutions:

• Paraffin sections, dry, prepared on Superfrost+ slides (Fisher, 12-550-15)
• CitriSolv (Fisher 04-355-12) or Xylene 
• PBST (PBS plus 0.05% Triton X)
• Antigen Retrieval Buffer (see recipe below)
• Goat Serum
• Primary antibody (NBP2-25158, Novus Biologicals)
• Secondary antibody (anti-chicken, such as Alexa Fluor 568, Goat polyclonal anti-Chicken IgY 
  (H and L) (Abcam, ab175477)
• Hydrophobic PAP pen (Abcam, ab2601)
• Vectashield with DAPI (Vector Laboratories, H-1200-10) or similar mountant

1. Deparaffinize: CitriSolv or Xylene (each batch fresh) 1x15 min, 1x15 min, 1x15 min

2. Rehydrate: 95% ethanol 2x5 min 

70% ethanol 1x5 min

PBS 3x5 min

3. Permeabilization: 0.05% PBST – 20 minutes at room temp

1. For 20 mls PBST: 10μl Triton X in 20 mls PBS

4. Antigen retrieval

*Pre-heat Antigen Retrieval Buffer in 95deg water bath 

1. Immerse section in retrieval buffer 95deg for 20 min
2. Cool to room temp

5. Block: 10% goat serum in 0.05% PBST – 1 hour at room temp

6. Primary Ab in PBST: anti-mCherry 1:200 – Overnight at 4°C. 

Keep slides horizontal by placing them on a covered slide rack. Use hydrophobic pen to make barrier around sections. Need ~150uL of antibody solution per slide. Add moistened paper towel to slide box to maintain humidity and help mitigate evaporation. 

7. Wash: PBST – 3 times, 5 minutes each (15 minutes total)

8. Secondary Ab in PBST: 1:500 – 45 minutes at room temp

9. Wash: PBST – 3 times, 5 minutes each (15 minutes total)

10. Coverslip with Vectashield/DAPI and image

­­

Antigen Retrieval buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0)

• Tri-sodium citrate (dihydrate) 2.94 g
• Distilled water 1 L
• Mix to dissolve. Adjust pH to 6.0 with 1M HCl.
• Add 0.5 mL Tween 20 and mix well. Store at room temperature or 4 deg for 3 months