ELISA Detection of Endogenous Serum Albumin in the Mouse Brain: A Measure of Extravasation Following Brain Injury

Materials and Reagents

1. Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories, catalog number: E90-134 )
   Note: This set includes an anti-mouse albumin goat antibody for plate coating, a mouse reference serum and a HRP-conjugated anti-mouse albumin goat antibody.
   
2. Mouse serum albumin (Sigma-Aldrich, catalog number: A3139 )
   
3. SureBlue^TM TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, catalog number: 52-00-01 )
   
4. 450 nm BioFX^® liquid Nova-Stop solution for TMB microwell substrates (SurModics, catalog number: LSTP-0100-01 )
   
5. BCA protein assay reagent A (Thermo Fisher Scientific, catalog number: 23221 )
   
6. BCA protein assay reagent B (Thermo Fisher Scientific, catalog number: 23224 )
   
7. Bovine serum albumin (BSA) set (Thermo Fisher Scientific, catalog number: 23208)
   
8. Pentobarbital sodium salt (Nacalai Tesque, catalog number: 26427-14 )
   
9. PBS (Santa Cruz Biotechnology, catalog number: sc-24946 )
   
10. Triton X-100 (Nacalai Tesque, catalog number: 35501-15 )
    
11. Tris (hydroxymethyl) aminomethane (Nacalai Tesque, catalog number: 35409-45 )
    
12. HCl (Nacalai Tesque, catalog number: 18321-05 )
    
13. NaCl (Nacalai Tesque, catalog number: 31320-05 )
    
14. NP-40 (Nacalai Tesque, catalog number: 23640-94 )
    
15. Deoxycholic acid (Wako Chemicals USA, catalog number: 044-18812 )
    
16. SDS (Nacalai Tesque, catalog number: 31607-65 )
    
17. EDTA (Nacalai Tesque, catalog number: 15114-15 )
    
18. Phenylmethylsulfonyl fluoride (Enzo Life Science, catalog number: ALX-270-184-G025 )
    
19. Aprotinin (Sigma-Aldrich, catalog number: A6103 )
    
20. PBS (see Recipes)
    
21. PBST (see Recipes)
    
22. Lysis buffer (see Recipes)
    

Equipment

1. Scissors (Fine Science Tools, catalog number: 14002-14 )
   
2. Forceps (Fine Science Tools, catalog number: 11271-30 )
   
3. Hemostats (Fine Science Tools, catalog number: 13002-10 )
   
4. Winged needle (Terumo, catalog number: SV-27DL )
   
5. Syringe (Terumo, catalog number: SS-30ESZ )
   
6. Razor (FEATHER Safety Razor, catalog number: FH-10 )
   
7. Acrylic brain slicer (Muromachi Kikai, catalog number: MK-MC-01 )
   
8. 1.5 ml sampling tubes (Bio-Bik, catalog number: RC-0170 )
   
9. 15 ml conical tube (BD Biosciences, Falcon^®, catalog number: REF 352196 )
   
10. 96-well plate (Thermo Fisher Scientific, catalog number: 167008 )
    
11. Pipette tips (2 to 200 µl) (Eppendorf, catalog number: 0030 073. 800 )
    
12. Stereotactic device (Narishige, model: SR-6N )
    
13. ELISA plate set (Sumitomo Bakelite, catalog number: BS-X7310 )
    Note: This set includes ELISA plates, plate seals, an antibody-immobilizing solution and a soaking solution.
    
14. Micropipettes (2 to 20 µl and 20 to 200 µl, Eppendorf)
    
15. Microplate reader (Bio-Rad Laboratories, model: 680 )
    
16. Digital Sonifier^® (Branson, model: 250D )
    
17. Centrifuge (Hitachi, model: CF15RXII )
    
18. Microplate mixer (AS ONE Corporation, model: NS-4P )
    

Procedure