166
YOSHITAKA HISHIKAWA ET AL.
11. Incubate with 40% deionized formamide/4x sse (over 30
mins) until used for hybridization.
Note: Deionized formamide (formamide 50 ml + Regin 2.5g):
Deionization should be immediately carried out for 30 mins.
Hybridization 12. Apply 30-40 f-ll of the hybridization mixture on each slide
and keep it in a moist chamber humidified with 40% formamide/4x sse at 37 - 42°e for 15-17 hrs.
Stringent
washing
munohistochemistry
Note: Before hybridization, the mixture is heat-denatured for 7
mins.
Note: The moist chamber should be completely shut offby masking tape.
13. Wash with 50% formamide/2x sSeI0.075% Brij 35 (3 x 1 hr)
at 37°e and with 2x sse (2 x 15 mins).
Note: For a more stringent washing 0.5 x sse and 0.2 x sse
should be used.
14. Apply 30-40 f-ll of the blocking solution and leave for 1 hr in a
moist chamber humidified with PBS.
Note: To minimize the extent of contaminsation with DNase and
RNase, care should be taken during handling of the specimens.
15. Apply 30-40 f-ll of the blocking solution containing HRP-labeled anti-(T-T dimer) monoclonal antibody (1:80) for 15-17
hrs in a moist chamber.
16. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each.
17. After washing with PBS, apply chromogen solution (DAB,
H20 2, nickel and cobalt) to the specimens and monitor
the reaction under a light microscope for optimum signal
and stop the reaction immediately by immersing in water
once the satisfactory color develops (Adams 1981).
18. Dehydrate with serial up-graded ethanol solutions, treat
with xylene and mount without any counterstaining.
YOSHITAKA HISHIKAWA ET AL.
11. Incubate with 40% deionized formamide/4x sse (over 30
mins) until used for hybridization.
Note: Deionized formamide (formamide 50 ml + Regin 2.5g):
Deionization should be immediately carried out for 30 mins.
Hybridization 12. Apply 30-40 f-ll of the hybridization mixture on each slide
and keep it in a moist chamber humidified with 40% formamide/4x sse at 37 - 42°e for 15-17 hrs.
Stringent
washing
munohistochemistry
Note: Before hybridization, the mixture is heat-denatured for 7
mins.
Note: The moist chamber should be completely shut offby masking tape.
13. Wash with 50% formamide/2x sSeI0.075% Brij 35 (3 x 1 hr)
at 37°e and with 2x sse (2 x 15 mins).
Note: For a more stringent washing 0.5 x sse and 0.2 x sse
should be used.
14. Apply 30-40 f-ll of the blocking solution and leave for 1 hr in a
moist chamber humidified with PBS.
Note: To minimize the extent of contaminsation with DNase and
RNase, care should be taken during handling of the specimens.
15. Apply 30-40 f-ll of the blocking solution containing HRP-labeled anti-(T-T dimer) monoclonal antibody (1:80) for 15-17
hrs in a moist chamber.
16. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each.
17. After washing with PBS, apply chromogen solution (DAB,
H20 2, nickel and cobalt) to the specimens and monitor
the reaction under a light microscope for optimum signal
and stop the reaction immediately by immersing in water
once the satisfactory color develops (Adams 1981).
18. Dehydrate with serial up-graded ethanol solutions, treat
with xylene and mount without any counterstaining.