178
YUKA IWASAKI AND IHACHI KA TO
In situ
hybridization
9. React the filter with a blocking solution for 1 hr at RT in a
vinyl bag. (250 J .. d/I-2 filters)
10. React the filter with the blocking solution including HRP-Iabeled sheep anti-digoxigenin antibody (1: 400-800) for 3 hrs
in a vinyl bag. (250 JlI/l-2 filters)
11. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each atRT.
12. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt at RT.
l3. Wash the filter at least 3 times with DDW for 5 mins each and
dry up.
We confirm the effectiveness of 28S r RN A staining as a reference
for optimization of the pretreatment conditions including protease digestion.
1. Cut sections to a thickness of 5-6 Jlm and place them on
silane-coated glass slides according to routine procedure.
2. Prior to use, warm at 60°C for at least 30 mins, deparaffinize
through toluene and then rehydrate with serial ethanol solutions and wash 3 times with PBS for 5 mins each.
3. Immerse in 0.3-3% H20 2 in methanol for 15 mins to inhibit
endogenous peroxidase activity, if necessary.
4. After a brief washing with PBS and DDW, immerse in 0.2 N
HCI for 20 mins and wash successively with DDW and PBS.
5. Treat with proteinase K (1-100 Jlg/ml in PBS) at 37°C for 10
mins and wash 3 times with PBS.
Note: Proteinase K solution is incubated in a water bath at 37°C
for 30 mins before this procedure. It is stored at -20°C in small
aliquots, which is used once and discarded. The effect of proteinase K digestion varies with the tissue and type of fixation,
so the concentration of proteinase K should be optimized
with individual experiment. In addition, the optimal concentration of proteinase K in the decalcified tissue sections (5-10 Jlg/
ml) is clearly lower than that for paraffin-embedded sections of
noncalcified tissue, i.e., 50-100 Jlg/ml.
YUKA IWASAKI AND IHACHI KA TO
In situ
hybridization
9. React the filter with a blocking solution for 1 hr at RT in a
vinyl bag. (250 J .. d/I-2 filters)
10. React the filter with the blocking solution including HRP-Iabeled sheep anti-digoxigenin antibody (1: 400-800) for 3 hrs
in a vinyl bag. (250 JlI/l-2 filters)
11. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each atRT.
12. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt at RT.
l3. Wash the filter at least 3 times with DDW for 5 mins each and
dry up.
We confirm the effectiveness of 28S r RN A staining as a reference
for optimization of the pretreatment conditions including protease digestion.
1. Cut sections to a thickness of 5-6 Jlm and place them on
silane-coated glass slides according to routine procedure.
2. Prior to use, warm at 60°C for at least 30 mins, deparaffinize
through toluene and then rehydrate with serial ethanol solutions and wash 3 times with PBS for 5 mins each.
3. Immerse in 0.3-3% H20 2 in methanol for 15 mins to inhibit
endogenous peroxidase activity, if necessary.
4. After a brief washing with PBS and DDW, immerse in 0.2 N
HCI for 20 mins and wash successively with DDW and PBS.
5. Treat with proteinase K (1-100 Jlg/ml in PBS) at 37°C for 10
mins and wash 3 times with PBS.
Note: Proteinase K solution is incubated in a water bath at 37°C
for 30 mins before this procedure. It is stored at -20°C in small
aliquots, which is used once and discarded. The effect of proteinase K digestion varies with the tissue and type of fixation,
so the concentration of proteinase K should be optimized
with individual experiment. In addition, the optimal concentration of proteinase K in the decalcified tissue sections (5-10 Jlg/
ml) is clearly lower than that for paraffin-embedded sections of
noncalcified tissue, i.e., 50-100 Jlg/ml.