1. 6-8 Lewis 20 5 4 2. (dark adaptation) 24 (5A) 3. 24 (6) 1, 6, 12, 24 800-900 lux (dark recovery) 24 (5B) 1. 6-8 Lewis 20 5 4 2. (dark adaptation) 24 (5A) 3. 24 1, 6, 12, 24 800-900 lux (dark recovery) 24 22
4. <1> 12 minocycline(100mg/kg) (7A) <2> minocycline(100mg/kg) (7B) <3> minocycline(100mg/kg) (8C) 1. Chlorohydrate 2. (OD) Davidson's solution4 24 3. (OS) -80 23
1. -80 200l PBS Solution (4, 5min, 13000R) 2. 200l Lysis buffer 100 3. 30 4. 30 (4, 15min, 13000R) BCA detergent-compatible (bicinchoninic aid; BCA) (quantitation) (colorimetric) (Cu 2 (Cu 1 ) (alkaline medium)[biuret reaction] (Cu 1 ) (Smith, PK et al., 1985) BCA 562 nm 20-2000g/ml BCA (end-point) 24
1. (BSA, Bovine Serum Albumin) (Working Range=20-2000g/ml) Volume of the BSA to Add Volume of Diluent to Add Final BSA Concentration 300l of (Stock) 0l 2000g/ml 375l of (Stock) 125l 1500g/ml(A) 325l of (Stock) 325l 1000g/ml(B) 175l of (A) 175l 750g/ml(C) 325l of (B) 325l 500g/ml(D) 325l of (D) 325l 250g/ml(E) 325l of (E) 325l 125g/ml(F) 100l of (F) 400l 25g/ml(G) 2. 25l dh 2 O microwell plate blank well 3. 5 25l (5l 20l dh 2 O ) microwell plate 4. standard solution(bsa) 25l 5. 200l working reagent (BCA Reagent A 3.6ml BCA Reagent B 72l ) microwell plate blank well 6. microwell plate 37 30 7. ELSA reader 562nm 8. 25
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) 112% Separation gel: H 2 O 4.3ml 40% Acrylamide mix (29:1) 3.0ml 1.5M Tris (ph8.8) 2.5ml 10% SDS 0.1ml 10% APS 0.1ml TEMED 0.004ml 2Stacking gel: H 2 O 2.87ml 40% Acrylamide mix (29:1) 0.5025ml 1.0M Tris (ph6.8) 0.5ml 10% SDS 0.04ml 10% APS 0.04ml TEMED 0.004 ml 1. 12% separation gel Hoefer minigel 26
separation gel isoproanol gel 2. separation gel isoproanol stacking gel running buffer 3. standard buffer sample buffer95 10 sample loading 4. 110V20-30mA 2 30 sample (Western blot) Mini Trans-blot Cell (Hoefer) ECL Western blotting detection reagents(amersham pharmacial biotech) X 1. SDS-PAGE 80V110mAOvernight PVDF membrane 2. PVDF membrane blocking buffer 30 27
3. PVDF membrane PBS solution 2 5 / 4. (Caspase-3PARP) (polyclonal Ab 11000) PVDF membrane 2 5. PVDF membrane PBS solution 3 5 / 6. (polyclonal Ab 1 1000) PVDF membrane 30 7. PVDF membrane PBS solution 1 5 / 8. PVDF membrane ECL Western blotting detection reagents 5 X 3-5 Hematoxylin & Eosin (Hematolxyin) Hematolxyin Harris's hematoxylin hematoxylin hemateinhematein 28
Hematoxylin (Eosin) (Eosin) hematoxylin H & E 1. 75 10 2. Xylene (99.8%, Merck) 9 3. 100 % Absolute ethanol 1 1 30 / 4. 90% ethanol 1 1 30 / 5. 80 % ethanol 1 1 30 / 6. dh 2 O 1 1 30 / 7. Hematoxylin 1 4 / 29
8. dh 2 O 1 1 30 / 9. 400 ml 75% ethanol (0.1% HCl 5 ml) 1 40 / 10. dh 2 O 1 5 / 11. Eosin 1 30 / 12. 80 % ethanol 1 1 10 / 13. 95 % ethanol 1 1 10 / 14. 100 % Absolute ethanol 1 1 50 / 15. Xylene 1 2 / 16. TUNEL(Terminal deoxynucleotidyl transferase-mediated dutp Nick End Labeling) TUNEL (apoptosis) DNA (necrosis) 30
ATP Apoptosis DNA 50 300 kb DNA DNA oligonucleosomal ladder(180bp ) (ladder pattern) 1992 Gavrieli terminal deoxynucleotidyl transferase(tdt) biotinylated deoxyuridine triphosphate(dutp) DNA 3'-OH TUNEL digoxigenin- nucleotide residues terminal deoynucleotidyl transferase(tdt) DNA TdT nucleotide triphosphate DNA 3'-OH Converter-POD (peroxidase) digoxigenin- nucleotide peroxidase DAB substrate (9) TUNEL 1. 75 10 2. Xylene (99.8%, Merck) 10 31
3. 100 % Absolute ethanol 1 3 / 4. 95% ethanol 1 3 / 5. 80 % ethanol 1 3 / 6. 70 % ethanol 1 3 / 7. PBS solution 1 3 / 8. dh 2 O 1 3 / 9. TUNEL 1. 300ml methanol(30%h 2 O 2 3ml)30 2. PBS solution5 1 3. 25l Working Strength TdT Enzyme(50l TdT Enzyme 450l Reaction buffer ) 4. 37 1 5. PBS solution15 1 6. 2% BSA (40mg BSA 2ml PBS ) 25l 15 7. PBS solution15 1 8. 25l Converter-POD 37 30 32
9. PBS solution15 1 10. 25l DAB Substrate Working Solution (20mg DAB, 0.005M Tris 50ml30% H 2 O 2 50l ) 10-20 11. dh 2 O 9 12. 70 % ethanol 1 1 / 13. 80 % ethanol 1 1 / 14. 95 % ethanol 1 1 / 15. 100 % Absolute ethanol 1 1 / 16. Xylene 1 2 / 17. 33