Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices

Materials and Reagents

1. Syringes (1 ml and 60 ml) and needles (24 and 27 gauge) (Becton Dickinson, catalog numbers: 309659 and 309653 )

   
2. Single edge razor blade (IDL TechniEdge, catalog number: 05-091C )

   
3. Double edge razor blade (Electron Microscopy Sciences, catalog number: 72002-10 )
   
4. Costar Netwell Insert with 440 μm polyester mesh (Corning, catalog number: 3478 )

   
5. Corning assay plate, black 384 well with non-binding surface (Corning, catalog number: 3575 )

   
6. Conical tube (15 ml) (BD Biosciences, catalog number: REF352196 )

   Note: Currently, it is “Thermo Fisher Scientific, Falcon^TM, catalog number: REF352196”.
   
7. Micro-centrifuge tubes (1.5 ml) (VWR International, catalog number: 20170-038 )

   
8. Aerosol barrier pipette tips (30 μl and 200 μl) (Thermo Fisher Scientific, catalog numbers: 02-707-33 and 02-707-42 )

   
9. Disposable transfer pipette (Thermo Fisher Scientific, catalog number: 13-711-9AM )

   
10. Glass plate (Electron Microscopy Sciences, catalog number: 70312-22 )

    
11. Glass staining dish (Newcomer supply, catalog number: 6816A )

    
12. Coarse fritted glass bubbler (Corning, catalog number: 39533-12EC )
    
13. Integrid Petri dishes (Becton Dickinson, Falcon, catalog number: 351112 )

    Note: Currently, it is “Thermo Fisher Scientific, Falcon^TM, catalog number: 351112 ”.
    
14. Adult (47-57 days old) male and female Sprague Dawley rats (Harlan Laboratories)
    
15. Hit-Hunter IP₃ fluorescence polarization assay kit (DiscoveRx Corporation, catalog number: 90-0037 )
    
16. 70% Perchloric acid (HClO₄) (Sigma-Aldrich, catalog number: 244252 )

    
17. Complete protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11697498001 )

    
18. Quick Start Bovine Serum Albumin (BSA) standard (Bio-Rad Laboratories, catalog number: 500-0206 )
    
19. Protein assay reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
    
20. Sodium pentobarbital solution (Virbac, catalog number: 710101 )
    
21. Calcium chloride solution (CaCl₂) (Sigma-Aldrich, catalog number: 21115 )

    
22. Magnesium chloride solution (MgCl₂) (Sigma-Aldrich, catalog number: M1028 )

    
23. Super glue (Super Glue Corporation, catalog number: SGP3 )

    
24. 70% ethanol (Thermo Fisher Scientific, Decon labs^TM, catalog number: 04-355-305 )
    
25. 95% O₂/5% CO₂ gas mixture (Airgas, catalog number: X02OX95C2003102 )
    
26. NaCl 

    
27. NaHCO₃
    
28. Dextrose
    
29. KCl
    
30. NaH₂PO₄
    
31. HEPES (pH 7.4)

    
32. EGTA

    
33. Sodium orthovanadate
    
34. NaF
    
35. Sodium pyrophosphate
    
36. PMSF
    
37. Artificial cerebrospinal fluid (aCSF) (see Recipes)
    
38. Sucrose aCSF (s-aCSF) (see Recipes)

    
39. HEPES homogenization buffer (see Recipes)

    

Equipment

1. Scissors (Fine science tools, catalog numbers: 14000-12 and 14060-10 )
   
2. Forceps (Stoelting, catalog numbers: 52104-37 and 52104-35 )

   
3. Hemostat (Fine science tools, catalog number: 13008-12 )

   
4. Spatula (Thermo Fisher Scientific, catalog number: S50822 )
   
5. Accuspin micro 17R microcentrifuge (Thermo Fisher Scientific, catalog number: 13-100-676 )

   
6. Biophotometer (Eppendorf AG, catalog number: 6133000010 )

   
7. Leica VT1200S vibrating blade microtome (Leica Biosystems Nussloch GmbH, catalog number: 1491200S001 )

   
8. Isotemp 105 water bath (Thermo Fisher Scientific, catalog number: S63077Q )

   
9. Qsonica Q500 sonicator (Thermo Fisher Scientific, catalog number: 15-338-282 )

   
10. Synergy 4 multimode microplate reader (BioTek Instruments)

    
11. Vapor pressure osmometer 5520 (Wescor)

    
12. Accumet AB15 plus pH meter (Thermo Fisher Scientific, catalog number: 13-636-AB15PC )
    
13. Rotator (Labline Instruments)

    
14. Micropipettes (2-20 μl, 20-200 μl and 100-1,000 μl) (Capitol Scientific, Eppendorf, catalog numbers: 3120000038 , 3120000054 and 3120000062 )

    

Procedure