Materials and Reagents

1. Milli-Q quality water (dH₂O)
   
2. Ethanol (Sigma-Aldrich, catalog number: 02854 )
   
3. Acetonitrile (Sigma-Aldrich, catalog number: 271004 )
   
4. K₂SO₄ (Merck KGaA, catalog number: 5153 )
   
5. NaCl (Merck KGaA, catalog number: 106404.1 )
   
6. CaCl₂^.2H₂O (CARLO ERBA Reagents, catalog number: 433381 )
   
7. NaN₃ (Merck KGaA, catalog number: 822335 )
   
8. NaOH (Sigma-Aldrich, catalog number: 221465 )
   
9. H₃PO₄ (Sigma-Aldrich, catalog number: P6560 )
   
   Enzymes
   
10. α-Amylase from porcine pancreas (Sigma-Aldrich, catalog number: A3176 )
    
11. Xylanase M1 from Trichoderma viride (Megazyme International, catalog number: E-XYTRI )
    
12. Lichenase [endo-1,3(4)-β-D-glucanase] from Bacillus sp. (Megazyme International, catalog number: E-LICHN )
    
    MALDI matrix preparation
    
13. 2,5-dihydroxybenzoic acid (DHB) (Sigma-Aldrich, catalog number: 85707 )
    
14. N,N-dimethylaniline (DMA) (analytical reagent grade) (Thermo Fisher Scientific, catalog number: 121-69-7 )
    
    Buffers and media
    
15. 70% ethanol (see Recipes)
    
16. 0.1 M NaOH (see Recipes)
    
17. 25 mM CaCl₂ (see Recipes)
    
18. Buffer for α-Amylase (see Recipes)
    
19. 1 mg/ml α-Amylase (see Recipes)
    
20. 460 U/ml Xylanase M1 (see Recipes)
    
21. 4.6 U/ml Xylanase M1 (see Recipes)
    
22. 200 U/ml Lichenase (see Recipes)
    
23. 2 U/ml Lichenase (see Recipes)
    
24. Saturated K₂SO₄ at 40 °C (see Recipes)
    
25. 50% acetonitrile (see Recipes)
    
26. MALDI matrix preparation (see Recipes)
    

Equipment

1. Vibratome (MICROM, model: HM 650 V )
   
2. In-house designed spraying robot
   Note: Robot was built by adapting an electro-spray probe dismounted from a LCQ Advantage mass spectrometer (Thermo Fisher Scientific) to an X, Y, Z robotic arm (FISNAR, F4300N) (see Figure 1) (see Note 2).
   
   
   Figure 1. In-house designed spraying robot for enzyme application. The right hand panel represents the enlarged view of the dashed rectangle zone depicted on the left picture.
   
   
3. Syringe pump infusion 0.2 µl/h to 500 ml/h (Thermo Fischer Scientific, catalog number: 12486350 )
   
4. 500 µl glass syringe (Hamilton, model: Gastight 1750 )
   
5. Thermo-shaker (Eppendorf, Thermomixer compact)
   
6. Incubator (THERMOSI, model: SR 300 )
   
7. Home-designed chamber for incubation
   Note: This consists of a rubber gasket sealed glass container (the jar with lid, KORKEN, IKEA of Sweden. Diameter 11 cm; high 10.5 cm, volume 0.5 L) in which is placed a 50 ml glass beaker and a set of weights (e.g. pieces of stones) (see Figure 2). Set of weights are required to keep the glass beaker from floating.
   
   
   Figure 2. Schematic representation of home-designed chamber for incubation
   
   
8. pH meter (pHenomenal^®, model: pH 1000 L , equipped with a pHenomenal^® 221 electrode)
   
   Other equipment
   
9. Glue (Loctite Super Glue Ultra Gel)
   
10. Adhesive carbon tape (8 mm x 20 m) (Agar Scientific, catalog number: AGG3939 )
    
11. Indium tin oxide (ITO) glass slides (Bruker, catalog number: 237001 ) (see Note 3)
    
12. Art paint brush (approximate diameter of the brush: 3-4 mm)
    
13. Razor blade (Gillette, Bleue extra)
    
14. Petri dish (90 mm x 14.2 mm) (Thermo Fischer Scientific, catalog number: 5184E )
    
15. Whatman filter paper (90 mm diameter)
    
16. 1.5 ml Eppendorf tube (Eppendorf Safe-Lock quality)
    
17. 2 ml plastic Pasteur pipette (Thermo Fischer Scientific, catalog number: 13984 )
    
18. Kimwipes paper (Kimberly-Clark)
    
    Additional, required for MALDI imaging experiments
    
19. ImagePrep nebulizing robot (Bruker Daltonics) (see Figure 3)
    
    
    Figure 3. ImagePrep nebulizing robot
    
    
20. MTP Slide adapter II (Bruker Daltonics, catalog number: 235380 ) (see Note 4)
    

Procedure