Iterative Indirect Immunofluorescence Imaging (4i) on Adherent Cells and Tissue Sections

Materials and reagents

For both tissue culture cells and FFPE tissue sections

1. L-Glycine-hydrochloride (Sigma-Aldrich, catalog number: G2879); storage: room temperature (RT)

2. Guanidium-hydrochloride (Sigma-Aldrich, catalog number: G3272); storage: RT

3. Urea (Sigma-Aldrich, catalog number: U5128); storage: RT

4. Tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich, catalog number: C4706); storage: 4 °C

5. N-acetyl-cysteine (Sigma-Aldrich, catalog number: A7250); storage: 4 °C

6. Bovine serum albumin (Sigma-Aldrich, catalog number: A9418); storage: 4 °C

7. Maleimide (Sigma-Aldrich, catalog number: 129585); storage: 4 °C

8. Phosphate-buffered saline (PBS) (Thermo Fisher, catalog number: 10010023); storage: RT

9. 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Thermo Fisher, catalog number: 15630080); storage: 4 °C

10. 12 M hydrochloric acid (Sigma-Aldrich, catalog number: 320331); storage: RT

11. 10 M sodium hydroxide (Sigma-Aldrich, catalog number: 72068); storage: RT

12. Primary antibodies (various manufacturers); storage: according to manufacturers’ recommendations

13. Secondary antibodies (Thermo Fisher, choose according to color requirements and species of primary antibodies. We recommend Donkey-anti-Primary antibodies); storage: according to manufacturers’ recommendations

14. 4’,6-Diamidino-2-phenylindole (Thermo Fisher, catalog number: 62247); storage: -20 °C

    
    

Specific for tissue culture cells

1. 96-well imaging plate (Greiner, catalog number: 655096); storage: RT

2. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710); storage: RT

3. Triton-X (Sigma-Aldrich, catalog number: X100); storage: RT

4. Succinimidyl Ester Alexa Fluor 647 (Thermo Fisher, catalog number: A20006)

   
   

Specific for FFPE tissue sections

1. Glass bottom Nexterion (Schott Glass, catalog number: 1535661)

2. Grace Bio-Labs ProPlate MP^TM microtiter plate superstructure (Sigma-Aldrich, catalog number: GBL204969-1EA)

3. National Diagnostics^TM Histo-Clear^TM tissue clearing agent (Chemie Brunschwig AG, catalog number: D1620333)

4. 100% EtOH (chemical under K20 chemical hood, was diluted in ddH₂O)

5. Feather A35 1 × 50 Blades (Biosystems Switzerland AG, catalog number: 81-0358-00); storage: RT

6. 30% w/v acrylamide/0.8% w/v bis-acrylamide (Sigma-Aldrich, catalog number: A3699-100ML); storage: 4 °C

7. TEMED (Thermo Fisher, catalog number: 17919); storage: 4 °C

8. Poly-L-lysine solution (Sigma-Aldrich, catalog number: P4832-50ML); storage: 4 °C

9. Glass basin length 21 cm × 15 cm × 6 cm

10. Superfrost plus microscope slides, 1 × 72 slides, 25 mm × 75 mm × 1 mm (Menzer-Glaseritem, catalog number: J1800AMNZ); storage: RT

11. PAP pen (Electron Microscopy Science, catalog number: 71310); storage: RT

12. Water-resistant, ethanol-soluble pen, 1 × 72 (Staedtler Lumocolor^® permanent pen 318)

13. Histoclear clearing agent (National Diagnostic, catalog number: HS-200)

    
    

Solutions

1. 40 mL elution buffer (EB) (see Recipes)

2. 6 mL 4i blocking buffer (sBB) (see Recipes)

3. 10 mL conventional blocking buffer (cBB) (see Recipes)

4. 20 mL 4i imaging buffer (IB) (see Recipes)

5. 20 mL fixation solution (FS) (see Recipes)

6. 10 mL permeabilization solution (PS) (see Recipes)

7. 5 mL primary antibody staining solution (1° AS) (see Recipes)

8. 5 mL secondary antibody staining solution (2° AS) (see Recipes)

9. 10 mL DNA stain solution (DSS) (see Recipes)

10. 10 mL whole protein staining solution (WPSS) (see Recipes)

11. Embedding solution (ES) (see Recipes)

Equipment

1. Microm HM355S (Thermo Scientific, catalog number: 90 520 0STS)

2. Steamer (Betty Bossi, catalog number: 10003614)

3. (Optional: to ease liquid handling steps) Washer dispenser (Agilent, catalog number: EL406)

Procedure