脑损伤后外渗的一种检测方法：酶联免疫吸附试验检测小鼠脑内血清白蛋白

Shotaro Michinaga; Yutaka Koyama

doi:10.21769/BioProtoc.1469

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Peer-reviewed

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

脑损伤后外渗的一种检测方法：酶联免疫吸附试验检测小鼠脑内血清白蛋白

Shotaro Michinaga

Yutaka Koyama email

发布: 2015年05月05日第5卷第9期 DOI: 10.21769/BioProtoc.1469 浏览次数: 10716

评审: Kae-Jiun ChangAnonymous reviewer(s)

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Jul 2014

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Abstract

After stroke and brain contusion, serum proteins extravasate into nerve tissue through disrupted blood-brain barrier (BBB). Because extravasations of serum proteins result in vasogenic brain edema, serum albumin level in the brain is an indicator of BBB disruption and brain edema after brain insults. In this protocol, extravasation of endogenous albumin is measured in the damaged mouse brain, which would be valuable in the evaluation of vasogenic brain edema formation (Michinaga et al., 2014).

Keywords: Blood-brain barrier (血脑障壁)

Brain edema (脑水肿)

Endothelial permeability (内皮通透性)

Brain microvessel (脑微血管)

Materials and Reagents

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.
Mouse serum albumin (Sigma-Aldrich, catalog number: A3139 )
SureBlue^TM TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, catalog number: 52-00-01 )
450 nm BioFX^® liquid Nova-Stop solution for TMB microwell substrates (SurModics, catalog number: LSTP-0100-01 )
BCA protein assay reagent A (Thermo Fisher Scientific, catalog number: 23221 )
BCA protein assay reagent B (Thermo Fisher Scientific, catalog number: 23224 )
Bovine serum albumin (BSA) set (Thermo Fisher Scientific, catalog number: 23208)
Pentobarbital sodium salt (Nacalai Tesque, catalog number: 26427-14 )
PBS (Santa Cruz Biotechnology, catalog number: sc-24946 )
Triton X-100 (Nacalai Tesque, catalog number: 35501-15 )
Tris (hydroxymethyl) aminomethane (Nacalai Tesque, catalog number: 35409-45 )
HCl (Nacalai Tesque, catalog number: 18321-05 )
NaCl (Nacalai Tesque, catalog number: 31320-05 )
NP-40 (Nacalai Tesque, catalog number: 23640-94 )
Deoxycholic acid (Wako Chemicals USA, catalog number: 044-18812 )
SDS (Nacalai Tesque, catalog number: 31607-65 )
EDTA (Nacalai Tesque, catalog number: 15114-15 )
Phenylmethylsulfonyl fluoride (Enzo Life Science, catalog number: ALX-270-184-G025 )
Aprotinin (Sigma-Aldrich, catalog number: A6103 )
PBS (see Recipes)
PBST (see Recipes)
Lysis buffer (see Recipes)

Equipment

Scissors (Fine Science Tools, catalog number: 14002-14 )
Forceps (Fine Science Tools, catalog number: 11271-30 )
Hemostats (Fine Science Tools, catalog number: 13002-10 )
Winged needle (Terumo, catalog number: SV-27DL )
Syringe (Terumo, catalog number: SS-30ESZ )
Razor (FEATHER Safety Razor, catalog number: FH-10 )
Acrylic brain slicer (Muromachi Kikai, catalog number: MK-MC-01 )
1.5 ml sampling tubes (Bio-Bik, catalog number: RC-0170 )
15 ml conical tube (BD Biosciences, Falcon^®, catalog number: REF 352196 )
96-well plate (Thermo Fisher Scientific, catalog number: 167008 )
Pipette tips (2 to 200 µl) (Eppendorf, catalog number: 0030 073. 800 )
Stereotactic device (Narishige, model: SR-6N )
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.
Micropipettes (2 to 20 µl and 20 to 200 µl, Eppendorf)
Microplate reader (Bio-Rad Laboratories, model: 680 )
Digital Sonifier^® (Branson, model: 250D )
Centrifuge (Hitachi, model: CF15RXII )
Microplate mixer (AS ONE Corporation, model: NS-4P )

Procedure

English

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Michinaga, S. and Koyama, Y. (2015). ELISA Detection of Endogenous Serum Albumin in the Mouse Brain: A Measure of Extravasation Following Brain Injury. Bio-protocol 5(9): e1469. DOI: 10.21769/BioProtoc.1469.