10-12 250-350 Sprague-Dawley rat ( SD ) 12 ( AM 8 00-PM 8 00 ) (Control group) (HS) (Heat shock group) 15 0 4 6 16 24 (PAE) ( Pulmonary air embolism group) 20 40 (25 l/min) 40 17
20 (HS&PAE) 15 4 16 20 40 (25µl /min) 40 20 / C/D (Compression/Decompression) 6 2 4 / HS C/D 4 4 18
(sodium pentobarbital) 50mg 41 15 4 6 16 24-70 (50 mg/kg) 0.58mm, 0.97mm polyethylene tube (PE50) 19
20 40 (25 l/min) (PE50) IL1610 Instrumentation Laboratory Milano Italy) 2.5 PBS (Dulbecco s Phosphate Buffered Saline) 20
4ml LDH (Lactate dehydrogenase LDH) / 60 0 C 48 LDH (830 x g 10 ) 20µl 0. 24mM NADH 500µl Tris/NaCl 9.8mM pyruvate 100µl (spectrophotometer) 339nm (830 x g 10 ), 50µl Coomassie Plus Protein Assay Reagent 1.5ml (spectrophotometer) 21
595nm (bovin serum albumin) 100µl 1% PMSF (phenylemthyl-sulfonyl fluoride) lysis buffer 12000 x g 4 10 10µl 20% BCA Plus Protein Assay Reagent (bovine serum albumin) BCA Plus Protein Assay Reagent (spectrophotometer) 562nm 22
SDS-PAGE (gel electrophoresis) (40µg) 95 0 C 5 0 0 C Acrylamide (stacking gel) (separating 20ml 10% running gel stacking gel stacking gel running buffer (comb) (well) 100 (transfer) (40µg) (comb) (well) 100 23
Coomassie brilliant blue R 250 (Western blotting) ( ) (Transfer) 2 nitrocellulose, Hybond-P; Amersham, membrane NC membrane 5 NC membrane 4 NC membrane NC membrane ( ) 70 PBST NC membrane NC membrane 5% (Anchor non-fat instant milk power, 24
New Zealand dairy board, New Zealand) PBST blocking buffer ( ) (4 0 C) blocking NC paper PBST NC paper 70 (HSP 70) (anti-hsp70,human (Mouse) monoclonal antibody) 2% PBST (112500) blocking buffer PBST IgG (goat anti-mouse IgG) PBST(112500) ECL (ECL kit, Amersham Inc,UK) X (Kodak) X. Eppendrof Centrifuge 5415R, Germany. Eppendrof Centrifuge 5417R, Germany. Eppendrof Centrifuge 5417C, Germany.. Misonix., Japan. 25
. Diagnostic & Research Instruments Co., Taiwan.. Shimadzu, UV-1601, Japan. TES Electrical Electronic Corp., Taiwan. Bio-Rad., U.S.A.. Bio-Rad., U.S.A.. Model 1000/500 Bio-Rad., U.S.A.. Instrumentation Laboratory IL 1610 ph/ Blood Gas Analyzer., Italy.. 1 (distilled water) 2 PMSF (phenylmethyl-sulfonyl fluride) Sigma Chemical 3 (BCA Plus Protein Assay Reagent)Pierce, U.S.A. 4 (bovine serum albumin) Bio-Rad., 26
U.S.A.. SDS-PAGE 1 running buffer 2 stacking gel solution 3 separating gel solution. 1 C.B.B (Coomassie brilliant blue R 250 ) Sigma Chemical Co. U.S.A. 2 (destain solution). (Western blotting) 1 (Filter paper) Advantec., Japan. 2 nitrocellulose, Hybond-P Amersham, Japan. 3 transfer buffer. immunochemical stain 1 New Zealand diary board, New Zealand. 2 Anti-HSP 70 monoclonal antibody Calbio chem., Germany. 27
3 Anti-mouse IgG (H+L) peroxidase from goat Jackson, U.S.A. 4 ECL Western blotting detection reagents Amersham, Germany.. 1 Sodium pentobarbital MTC, Canada. 2 30% Acrylamide / Bis Solution Bio-Rad., U.S.A. 3 Ammonium Persulfate (APS) Bio-Rad., U.S.A. 4 Antifoam A Sigma, U.S.A. 5 Aprotinin Sigma, U.S.A. 6 Bromophenol Blue Sigma, U.S.A. 7 Chymostain Sigma, U.S.A. 8 Dulbecco s Phosphate Buffered Saline PBS Life Techologies, U.S.A. 9 Glycline Sigma, U.S.A. 10 Methanol Merck, Gemany. 11 Pepstatin A Sigma, U.S.A. 12 Potassium Chloride KCl Sigma, U.S.A. 28
13 Potassium Phosphate Dibasic K 2 HPO 4 Sigma, U.S.A. 14 Potassium Phosphate KH 2 PO 4 Sigma, U.S.A. 15 Sodium dodecyl sulfate SDS Sigma, U.S.A. 16 Sodium Chloride Sigma, U.S.A. 17 Tris Bio-Ras, U.S.A. 18 TEMED Sigma, U.S.A. 19 Tween-20 Sigma, U.S.A. 20 Thimerosal Sigma, U.S.A. 21 Trition X-100 Sigma, U.S.A. 22 2-Mercaptoethanol Sigma, U.S.A. (mean SD) two-way ANOVA with repeated measurement P < 0.05 one-way ANOVA P < 0.05 29