Materials and Reagents

1. Sterile disposable pipettes (10 ml and 5 ml)
   
2. Aluminum foil
   
3. Four well tissue culture plates (Thermo Fisher Scientific, Nunc^TM, catalog number: 176740 )
   
4. Round Glass coverslips (12 mm) (Bellco Glass, catalog number: 1943-10012A )
   
5. Parafilm
   
6. Entamoeba histolytica HM1:IMSS strain (a kind gift from Prof. Alok Bhattacharya, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India)
   
7. Fluorescently labeled fibronectin (CYTOSKELETON, catalog number: FNR02 )
   
8. Glutaraldehyde (25% stock solution in dH₂O, EM grade) (Sigma-Aldrich, catalog number: G5882 )
   
9. Sodium Borohydride (NaBH₄) (Sigma-Aldrich, catalog number: 452882 )
   
10. Poly-L-lysine (Sigma-Aldrich, catalog number: P8920 )
    
11. 70% ethanol (Merck Millipore Corporation)
    
12. Alexa 568 Phalloidin (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: A12380 )
    
13. 1% BSA (Sigma-Aldrich, catalog number: A-7906 ) in PBS
    
14. Mowiol mounting medium (Sigma-Aldrich, catalog number: 81381 )
    
15. Complete BI medium (incomplete supplemented with 15%, Adult Bovine Serum)
    
16. GasPak^TM EZ Anaerobe Pouch System (BD Biosciences, catalog number: 260683 )
    
17. Sterile dH₂O
    
18. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    
19. KCl
    
20. Potassium phosphate monobasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: P5655 )
    
21. Potassium phosphate dibasic trihydrate (KH₂PO₄) (Sigma-Aldrich, catalog number: P5504 )
    
22. NaOH
    
23. Hilyte 488 Fibronectin
    
24. Biosate peptone BBL (BD Biosciences, catalog number: 211862 )
    
25. Glucose (Dextrose) (Sigma-Aldrich, catalog number: D9434 )
    
26. L-cysteine (Sigma-Aldrich, catalog number: C1276 )
    
27. L-ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
    
28. Ferric ammonium citrate (Sigma-Aldrich, catalog number: F5879 )
    
29. 10x phosphate-buffered saline (PBS) (see Recipes)
    
30. 0.01% poly-L-lysine solution (see Recipes)
    
31. 0.5% glutaraldehyde solution (see Recipes)
    
32. 5 mg/ml sodium borohydride (see Recipes)
    
33. 4% paraformaldehyde (PFA) (see Recipes)
    
34. 1% BSA in PBS (see Recipes)
    
35. 0.1% Triton-X-100 (see Recipes)
    
36. 100 μg/ml Hilyte488 Fibronectin (FNR02) (see Recipes)
    
37. Basal incomplete medium (see Recipes)
    
38. Complete medium (see Recipes)
    

Equipment

1. Laminar air flow hood
   
2. 37 °C incubator
   
3. Forceps
   
4. Moist chamber
   
5. Vacuum Pump and glass Pasteur pipettes with tubings
   
6. Table top centrifuge with swinging bucket rotor
   

Procedure