*Genetic monitoring protocol

 

Table. Biochemical markers frequently used for genetic monitoring

Locus symbol and name

Allels

Chromosome No.

Akp-1

Alkaline phophatase-1

a, b

1

Car-2

Carbonic anhydrase-2

a, b

3

Ce-2

Kidney catalase-2

a, b

17

Es-1

Esterase-1

a, b

8

Es-2

Esterase-2

a, b, c

8

Es-3

Esterase-3

a, b, c

11

Es-10

Esterase-10

a, b, c

14

Gpd-1

Glucose-6-phophate dehydrogenase-1

a, b, c

4

Gpi-1

Glucose phosphate isomerase-1

a, b

7

Hbb

Hemoglobin beta-chain

d, p, s

7

Idh-1

Isocitrate dehydrogenase-1

a, b

1

Ldr-1

Lactate dehydrogenase regulator

a, b

6

Mod-1

Malic enzyme supernant

a, b

9

Mup-1

Major urinary protein

a, b

4

Pep-3

Peptidase-3

a, b, c

1

Pgm-1

Phosphoglucomutase-1

a, b

5

Trf

Transferrin

a, b

9

 

 

 

 

 

 

 

1. Alkaline phosphatage-1 (Akp-1 Chromosome No.1)

 Tissue sample: Kidney homogenates 0.6

 Buffer system: Tris citrate; pH 8.2

                Tris

10.5g

citric acid

3.0g

Bring up to lwith D.W.

 

 Electrophoresis: 200 volts , 40 minutes, Cathode (-) to Anode (+)

 Stain procedure: (a) 50 mg beta-naphthyl-phosporic acid

                     5 ml distilled water

                 (b) 50 mg fast blue RR salt

                     10 mg MgCl₂ㆍ6H2O

                     10 mg MnCl₂ㆍ4H2O

                     5 ml tris HCl(pH 9.0:Tris 24.2g in 800, pH 맞춘후 1ℓ만듦.)

                Mix (a) and (b) just before staining

                Incubate at 37 for 20 minutes

                Fix in 5% acetic acid solution

 Type strain: Akp-1A: C57BL/6

             Akp-1B: C3H/He

 

 

2. Carbonic anhydrase-2 (Car-2 Chromosome No.3)

   Tissue sample: RBCs in 3volume D.W.  0.6

   Buffer system: EDTA sodium acetate; pH 5.6

                  NaC2H3·3H2O

17.01g

EDTA(tetrasodium salt)     

2.48g

Bring up to lwith D.W.

   Electophoresis: 120 volts, 60 minutes , Anode (+) to Cathode (-)

   Stain procedure: Apply 0.5% Ponceau S in 10% sulfosalicylic acid

                   Destain in 5% acetic acid

   Type strain: Car-2A: C57BL/6

                Car-2B: DBA/2

 

 

3. Kidney catalase-2 (Ce-2 Chromosome No.17)

(1) Method 1

   Tissue sample: Kidney homogenates in 10 weight distilled water

                  Let sit 30 minutes , Dilute 1:1 before application

   Buffer system: Tris citrate; pH 8.6

                  Tris           

9.25g

Citric acid

10.5g

Bring up to lwith D.W.

Adjust to up pH 8.6 with 1M citric acid

   Electrophoresis: 350 volts, 15 minutes, Cathode (-) to anode (+)

   Stain procedure: Flood with 0.06% hydrogen peroxid for 1 minute and rinse with water. After blotting apply ferricyanide stain (1% ferric chloride:1% potassium ferricyanide=1:1) until bands appear.

                    Flood with water

   Type strain: Ce-2A: C57BL/6

               Ce-2B: C3H/He

 

 

 (2) Method 2

   Tissue sample: Kidney homogenates in 10 weight D.W.

   Buffer system: Tris glycine: pH 8.5 

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts , 25 minutes, cathode (-) to anode (+)

   Stain procedure: Flood with 0.03% hydrogen peroxide for 30 seconds and rinse with water. Apply ferricanide stain (1% ferric chloride: 1% potassium ferricyanide=1:1)

   Type strain: Ce-2A: C57BL/6

               Ce-2B: C3H/He

 

4. Esterase-1 (Es-1 Chromosome No.8)

  Tissue sample: Plasma  0.3

  Buffer system: 0.01M Phosphate; pH 6.8

                KH2PO4

3.77g

NaHPO4·12H2O

8.63g

Bring up to lwith D.W

  Electrophoresis: 140 volts ,30minutes, Cathode (-) to anode (+)

  Stain procedure:

       0.5 ml 2% beta-naphthyl acetate (2g beta-naphthyl acetate in 100 acetone)

       50 mg fast blue RR salt

       10 ml 0.01M phosphate; pH 6.8

       Incubate at 37 until bands appear

       Fix in 5% acetic acid

  Type strain: Es-1A: C57BL/6

              Es-1B: DBA/2

 

5. Esterase-2 (Es-2 Chromosome No.8)

  Tissue sample: Kidney homogenates in 5 weight distilleld water 0.6

  Buffer system: 0.01M Phosphate; pH 6.8

                KH2PO4

3.77g

NaHPO4·12H2O

8.63g

Bring up to lwith D.W

  Electrophoresis: 140 volts , 30 minutes, Cathode (-) to anode (+)

  Stain procedure:

        0.5 ml 2% beta-naphthyl acetate(2g beta-naphthyl acetate in 100 acetone)

        50 mg fast blue RR salt

        10 ml 0.01M Phosphate; pH 6.8

        Incubate at 37 until bands appear

        Fix in 5% acetic acid

  Type strain: Es-2A: KK

               Es-2B: C57BL/6

               Es-2C PL/J

 

 

6. Esterase-3 (Es-3 Chromosome No.11)

  Tissue sample: Kidney homogenates in 5 weight D.W. 0.6

  Buffer system: Tris EDTA borate; pH 8.4

                Tris

10.91g

EDTA(disodium salt)

0.6g

Boric acid

3.1g

Bring up to lwith D.W

 

  Electrophoresis: 350 volts, 20 minutes, Cathode (-) to anode (+)

  Stain procedure:

        0.5 ml 2% beta-naphthyl acetate(2g beta-naphthyl acetate in 100 acetone)

        50 mg fast blue RR salt

        10 ml 0.01M Phosphate; pH 6.8

        Incubate at 37 until bands appear

        Fix in 5% acetic acid

  Type strain: Es-3A: C57BL/6

               Es-3B: RF/J

               Es-3C: DBA/2

 

 

7. Esterase-10 (Es-10 Chromosome No.14)

  Tissue sample: RBCs in 3 volumes D.W.

  Buffer system: Tris glycine; pH 8.9

                Tris

5.16g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

  Electrophoresis: 300 volts, 25 minutes, Cathode(-) to anode (+)

  Stain procedure: Flood with fluorescent stain

                     4-methyl-umbelliferyl acetate

3.0mg

Acetone

0.1

0.05M Potassium phosphate monobasic pH 7.0

5

                              Incubate 15 minutes at 37

                  Read under UV light

  Type strain: Es-10A: C57BL/6

               Es-10B: DBA/2

               Es-10C: BUB/BnJ

 

 

8. Glucose-6-phosphate dehydrogenase-1 (Gpd-1 Chromosome No.4)

 

 (1) Method 1

   Tissue sample: Kidney homogenates in 5 weight D.W. 0.6

   Buffer system: Tris EDTA borate; pH 8.4

                 Tris

10.91g

EDTA(disodium salt)

0.6g

Boric acid

3.1g

Bring up to lwith D.W

   Electrophoresis: 350 volts, 20 to 30 minutes, Cathode(-) to anode (+)

   Stain procedure: 20mg glucose-6-phosphate

                   4mg MTT

                   2mg PMS

                   8mg NADP+

                   5mg MgCl₂ㆍ6H2O

                   10ml Tris-HCL; pH 7.0

                     (24.2g Tris in 800 D.W. After adjust pH bring up to 1)

                   Incubate at 37 until bands appear

                   Fix in 5% acetic acid

   Type strain: Gpd-1A: C57BL/6

               Gpd-1b: DBA/2

               Gpd-1c: Several wild mice             

 

 

 

 

 (2) Method 2

   Tissue sample: Liver homogenates in 10 weight D.W.

                  Add 0.04-0.08 NADP (10mg/ml)

   Buffer system: Tris glycine; pH 8.9

                 Tris

5.16g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts, 60 minutes, Cathode(-) to anode (+)

   Stain procedure: In agar overday:

                  3ml 1% molten agar (Cool to 56 before use)

                  2ml Tris-HCL; pH 8.0

                  0.12ml 0.25M  Mg(C2H3O2)2

                  0.12ml NADP (10 mg/ml)

                  0.12ml 0.008M PMS

                  0.12ml 0.02M MTT

                  0.24ml 0.5M G-6-P

   Type strain: Gpd-1A: C57BL/6

               Gpd-1b: DBA/2

               Gpd-1c: Several wild mice

 

9. Glucose phosphate isomerase-1 (Gpi-1 Chromosome No.7)

 

(1) Method 1

   Tissue sample: Kidney homogenates in 5 weight D.W.

                  Dilute 1:10 with distilled water. 0.3

   Buffer system: 0.01M Phosphate; pH 6.8

                  KH2PO4

3.77g

NaHPO4·12H2O

8.63g

Bring up to lwith D.W

   Electrophoresis: 160 volts, 60 minutes, Cathode(-) to anode (+)

   Stain procedure: 20mg fructose-6-phosphate

                   4mg MTT

                   2mg PMS

                   8mg NADP+

                   20 units G-6-PD

                   16mg MgCl₂ㆍ6H2O

                   10ml Tris-HCL; pH 8.0

                       (24.2g Tris in 800 D.W. After adjust pH bring up to 1)

                   Incubate at 37 until bands appear

                   Fix in 5% acetic acid

   Type strain: Gpi-1A : DBA/2

                Gpi-1B : C57BL/6

 

 

 

 

 

 

 

 

(2) Method 2

   Tissue sample: RBCs in 3 volumes D.W. or Kidney in 10 weight D.W. or

                  Liver in 10 weight D.W.

   Buffer system: Tris glycine; pH 8.5

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts, 30 minutes, Cathode(-) to anode (+)

   Stain procedure:  In agar overday:

                  9ml 1% molten agar(Cool to 56 before use)

                  2ml Tris-HCL; pH 8.0

                     (24.2g Tris in 800 D.W. After adjust pH bring up to 1)

                  0.12ml 0.25M  Mg(C2H3O2)2

                  0.12ml NADP (10 mg/ml)

                  0.12ml 0.008M PMS

                  0.12ml 0.02M MTT

                  0.12ml 10% F-6-P

                  0.003 ml G-6-PD (about one enzyme unit)

   Type strain: Gpi-1A : DBA/2

                Gpi-1B : C57BL/6

 

 

10. Hemoglobin beta-chain (Hbb Chromosome No.7)

 

  (1) Method 1

   Tissue sample: RBCs in 3 volumes D.W. 0.3

   Buffer system: Tris EDTA borate; pH 8.4

                 Tris

10.91g

EDTA(disodium salt)

0.6g

Boric acid

3.1g

Bring up to lwith D.W

   Electrophoresis: 350 volts, 30 minutes, Cathode(-) to anode (+)

   Stain procedure: Apply 0.5% Ponceau S in 10% sulfosalicylic acid

                   Destain in 5% acetic acid

   Type strain: HbbD: DBA/2

                HbbP: AU/SsJ

                HbbS: C57BL/6

 

 

 (2) Method 2 (Hbb)

   Tissue sample: RBCs in 20 volumes D.W.

   Buffer system: Tris glycine; pH 8.9

                 Tris

5.16g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 350 volts, 12 minutes, Cathode(-) to anode (+)

   Stain procedure: 0.02g ο-dianisidine

                   0.5ml glacial acetic acid

                   0.7 ml 3% H2O2

                                       Flood plate until bands turn green

                   Rinse with water

   Type strain:  HbbD: DBA/2

                HbbP: AU/SsJ

                HbbS: C57BL/6

 

 

 (3) Method 3 (Hbb)

   Tissue sample: RBCs in 3 volumes D.W.

   Buffer system: Tris glycine; pH 8.5

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts, 30 minutes, Cathode(-) to anode (+)

   Stain procedure: Apply 0.5% Ponceau S in 5% trichliri-acetic acid

                   Destain in 5% acetic acid

 

 

 

11. Isocitrate dehydrogenase-1 (Idh-1 Chromosome No. 1)

 

(1) Method 1

   Tissue sample: Kidney in 5 D.W. 0.6

   Buffer system: Tris citrate; pH 8.6

                 Tris

9.25g

Citric acid

10.5g

bring up to 1with D.W.

adjust to pH 6.8 with 1M citric acid

   Electrophoresis: 200 volts, 40 minutes, Cathode(-) to anode (+)

   Stain procedure: 4mg NBT

                   3mg PMS

                   4mg NADP+

                   40mg DL- isocitrate (trisodium salt)

                   50mg MnCl₂ㆍ4H2O

                   10ml distilled water

                   Incubate at 37 until bands appear

                   Fix in 5% acetic acid

    Type strain: Idh-1A: C57BL/6

                Idh-1B: DBA/2

 

  (2) Method 2

   Tissue sample: Kidney homogenates in 10 weight D.W.

   Buffer system: Tris glycine; pH 8.5

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts, 30 minutes, Cathode(-) to anode (+)

   Stain procedure:  In agar overday:

                  3ml 1% molten agar (Cool to 56 before use)

                  2ml Tris-HCL; pH8.0

                      (24.2g Tris in 800 D.W. After adjust pH bring up to 1)

                  0.04ml 0.1M MnCl

                  0.12ml NADP (10 mg/ml)

                  0.12ml 0.008M PMS

                  0.12ml 0.02M MTT

                  0.048ml 10% isocitrate (trisodium salt)

   Type strain: Idh-1A: C57BL/6

                Idh-1B: DBA/2

 

 

12. Lactate dehydrogenase regulator (Ldh-1 Chromosome No. 6)

  Tissue sample: RBCs in 3 volumes D.W. 0.6

  Buffer system: Tris citrate; pH 8.3

                Tris

16.64g

Citric acid

4.2g

bring up to 1 with D.W.

 

 

  Electrophoresis: 200 volts, 40 minutes, Cathode(-) to anode (+)

  Stain procedure:

      1ml 1M  NaDL-lactate

         (10.6ml 85% DL-lactate, 6.1g Na2CO3H2O, Bring up to 100ml with water)

      2mg NBT

      1mg PMS

      2mg NAD+

      0.5ml 0.1M NaCN

      10ml Tris-HCL; pH 7.0

         (24.2g Tris in 800 D.W. After adjust pH bring up to 1)

      Incubate at 37 until bands appear

      Fix in 5% acetic acid

  Type strain: Ldr-1A: C57BL/6

              Ldr-1B: LP/J, DW/J

 

13. Malic enzyme supernatant (Mod-1 Chromosome No. 9)

 

 (1) Method 1

   Tissue sample: Kidney homogenates in 10weight D.W.

   Buffer system: Tris citrate; pH 7.6

                 Tris

12.1g

D.W.

600

Adjust to pH 7.6 with 10% citric acid

Bring up to 1 with D.W.

   Electrophoresis: 200 volts,  30 minutes, Cathode(-) to anode (+)

   Stain procedure: In agar overday:

         3ml 1% molten agar(Cool to 56 before use)

         2ml 0.2M Tris-HCL; pH 8.0

              (24.2g Tris in 800 D.W. After adjust pH bring up to 1)

         0.12ml 0.01M MnCl

         0.12ml NADP (10 mg/ml)

         0.12ml 0.008M PMS

         0.12ml 0.02M MTT

         0.48ml 0.5M malate

                  Malic acid in 10 D.W.

2.66g

Adjust to pH 8.0 with NaOH

Dilute to 40 with D.W.

         Incubate at 37 until bands appear

   Type strain: Mod-1A: DBA/2

                Mod-1B: C57BL/6

 

 

 (2) Method 2

   Tissue sample: Kidney homogenates in 10 weight D.W.

   Buffer system: Tris EDTA borate; pH 9.1

                 Tris

10.53g

EDTA(disodium salt)

0.92g

Boric acid

0.54g

Bring up to 1 with D.W.

   Electrophoresis: 350volts, 12 minutes, Cathode(-) to anode (+)

   Stain procedure: In agar overday:

                  10ml 1% molten agar (Cool to 56 before use)

                  0.025ml 0.07M MnCl

                  0.1ml NADP (10 mg/ml)

                  0.1ml 0.008M PMS

                  0.1ml 0.02M MTT

                  0.5ml 0.5M malate

                      Malic acid in 10 D.W.

2.66g

Adjust to pH8.0 with NaOH

Dilute to 40 with D.W.

                    

                  Incubate at 37 until bands appear

   Type strain: Mod-1A: DBA/2

                Mod-1B: C57BL/6

 

 

 

 

 

 

 

14. Major urinary protein (Mup-1 Chromosome No. 4)

   Tissue sample: Urine in same volume D.W.

   Buffer system: Tris glycine; pH 8.5

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 150 volts, 20 minutes, Cathode(-) to anode (+)

   Stain procedure: Apply 5% Ponceau S in 5% trichloro-acetic acid

                   Destain in 5% acetic acid

   Type strain: Mup-1A: DBA/2

               Mup-1B: C57BL/6

 

 

15. Peptidase-3 (Pep-3 Chromosome No. 1)

 

 (1) Method 1

   Tissue sample: Kidney homogenates 10 weight D.W.

   Buffer system: Tris glycine; pH 8.5

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts, 25 to 30minutes, Cathode(-) to anode (+)

   Stain procedure: In agar overday:

                  3ml  molten agar (Cool to 56 before use)

                  1ml 0.2M phosphate; pH 6.8

                      KH2PO4

27.2g

D.W.

800

Adjust to pH 6.8 with 6N NaOH

Bring up to 1 D.W.

                  1ml distilleld water 

                  0.04ml Crotalus adamanteus venom (2.5mg/ml)

                  0.04ml peroxidase (10mg/ml)

                  0.04ml L-leucyl-L-tyrosine (10mg/ml)

                  0.08ml 1% ο-dianisidine

                  Incubate at 37 until bands appear

   Type strain: Pep-3A: C57BL/6

                Pep-3B: DBA/2

                Pep-3C: NZB,DD

 

 

 

 

 

 

 

 

 

 (2) Method 2

   Tissue sample: Kidney homogenates in 5 weight D.W. 0.3

   Buffer system: Tris EDTA borate; pH 8.4

                 Tris

10.93g

EDTA(disodium salt)

0.6g

Boric acid

3.1g

Bring up to 1 with D.W.

   Electrophoresis: 300 volts, 20 minutes, Cathode(-) to anode (+)

   Stain procedure: 20mg L-leucyl-alanine

                   5mg MTT

                   5mg PMS

                   5mg L-amino acid oxidase

                   5mg peroxidase

                   10ml 0.01M phosphate; pH 6.8 (1:15 diluted 0.01M phosphate)

                   Incubate at 37 until bands appear

                   Fix in 5% acetic acid

   Type strain: Pep-3A: C57BL/6

                Pep-3B: DBA/2

                Pep-3C: NZB,DD

 

 

16. Phosphoglucomutase-1 (Pgm-1 Chromosome No. 5)

   Tissue sample: Kidney/liver homogentes in 10 weight D.W. or RBCs

   Buffer system: Tris glycine; pH 8.5

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts, 45 minutes, Cathode(-) to anode (+)

   Stain procedure: In agar overday:

                  3ml 1% molten agar  (Cool to 56 before use)

                  2ml  Tris-HCL; pH 8.0 (B22)

                       Tris

24.2g

D.W.

800

Adjust pH with concentrated HCl

Bring up to 1 with D.W.

                  0.16ml 0.02M MTT

                  0.16ml 0.008M PMS

                  0.16ml NADP (10 mg/ml)

                  0.02ml G-1, 6-DP(10mg/ml)

                  0.005ml G-6-PD (300 units/ml)

   Type strain: Pgm-1A: C57BL/6

                Pgm-1A: DBA/2

 

 

 

 

 

 

17. Transferrin (Trf Chromosome No. 9)

 

 (1) Method 1

   Tissue sample: Plasma 0.6

   Buffer system: Tris borate; pH 8.4

                 Tris

24.0g

Boric acid

12.0g

Bring up to 1 with D.W.

   Electrophoresis: 400 volts, 30 minutes, Cathode(-) to anode (+)

   Stain procedure: Apply 0.5% Ponceau S in 10% sulfosalicylic acid

                   Destain in 5% acetic acid

   Type strain: TrfA: CBA

                TrfB: C57BL/6

 

 (2) Method 2

   Tissue sample: Plasma  0.6

   Buffer system: Tris glycine; pH 6.5

                 Tris

3.0g

Glycine(NH3 free)

14.4g

Bring up to lwith D.W.

   Electrophoresis: 200 volts, 25 minutes, Cathode(-) to anode (+)

   Stain procedure: Apply 0.5% Ponceau S in 5% trichloro-acetic acid

                   Destain in 1% acetic acid

   Type strain: TrfA: CBA

                TrfB: C57BL/6

 

유전 모니터닝

 

1. Cellulose acetate menabrane 준비

1.1. 완충액(Buffer)을 준비한다. 이 완충액을 비이커에 300 붓는다.

주의: 완충액 제조시 용량을 정확하게 한다. 틀릴 경우 영동결과에 큰 영향을 나타낸다.

 

1.2. Cellulose acetate menabrane를 조심스럽게 잡고 플라스틱면에 필요한 사항을 기입한다.

주의: 막면에 손이 닿지 않도록 글러브를 착용한다.

 

1.3. 천천히 완충액에 Cellulose acetate menabrane을 넣고 5분간 침적한다.

주의: 급속히 넣으면 막면에 기포가 생기기 쉽다.

주의: 완충액에 넣은 막은 1-2일내에 사용한다.

 

2. 전기영동준비

2.1. 플라스극과 마이너스극의 전극 브릿지에 전극여지를 걸친다.

주의: 브릿지를 따라 손가락 끝으로 여지를 밀착시킨다.

 

2.2. 완충액을 각각의 완충액 용기에 넣고 약 60씩을 양 전극조에 넣는다.

주의: 기포가 있는 경우 손가락으로 제거한다.

주의: 여지가 완충액에 충분히 침적한 것을 확인한다.

 

3. 전기영동

3.1. 시료 30㎕를 샘플 well에 분주한다.

주의: 슬릿트에 생긴기포는 가능하면 없앤다.

주의: 사용중에 시료를 냉장보관한다.

 

3.2. 완충액에 침적해두었던 Cellulose acetate menabrane을 꺼내어 킴스타월 사이에 넣어 여분의 완충액을 흡수시킨다. 막면을 위로해둔다.

주의: 이 이후의 조작은 신속하게 한다.

 

3.3. 막의 끝으로부터 2㎝의 선에 샘플 comb으로 시료를 살짝 찍는다.

주의: 샘플 comb선단의 슬릿트에 시료가 균일하게 들어가 있는 것을 확인한다.

 

3.4. 시료를 적용한 쪽을 마이너스극 쪽에 오도록 하고 막 면은 아래로 하여 chamber(영동조)에 넣는다.

 

3.5. 뚜껑을 덮고 막 고정장치를 내려 고정한다.

 

3.6. 전원을 영동장치에 연결한다.

 

4. 염색

4.1. 영동 종료후 Cellulose acetate menabrane의 막면을 아래로 하여 앰색액에 넣는다.

주의: 염색전에는 막이 건조되지 않도록 한다.

주의: 막을 염색액에 뜨도록 놔두어도 무방하다.

 

4.2. 20초후 염색용기를 기울여 여분의 염색액을 제거한다. 킴스페이퍼를 막을 덮고 막전체를 위로부터 누르면서 여분의 염색액을 제거한다.

주의: 염색액은 폐액을 취급한다.

 

4.3. 37℃에 incubation

주의: 반응을 빈번히 관찰한다. 반응이 지나치지 않도록 주의한다.

 

4.4. 5% 초산 용액(acetic acid soln.)을 만든다. Cellulose acetate menabrane이 들어있는 용기에 적당량을 넣는다.

주의: 처리시간은 5분정도가 적당하다.

 

4.5. 결과를 판정한후 비닐에 넣어 밀봉, 보존한다.

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Lab Anim Med, College of Vet Med, Seoul Nat'l Univ, Sinlim, Kwanak, Seoul, Korea 151-742