All solutions and containers must be RNase-free.
200-250 ml fills a baked glass dish.
I. Tissue sections.
Pre-warm slides of frozen sections at RT for at least 30 mins.
II. Treatment of the slides before hybridization.
1) Fix slides in 4%PFA/PBS for 10 min, RT.
2) Wash 3 x 3 min with PBS, each time transfer to a new dish.
3) Treat with TE/proteinase K, 7 mins (no more):
Tris 1M, pH 7.5 DEPC, 12. 5 ml
EDTA 500 mM, 2.5 ml
adjust volume to 250 ml, add 375 μl pro K
4) Put slides back in 4%PFA/PBS for 5 min, then 5 min PBS.
5) Set up a dish with a stir bar and 2 blue tips to hold slide tray above
stir bar. Quickly make a solution of 4.63g triethanolamine, 0.56 ml 10N
NaOH in 250 ml H20, and pour it into the dish. Set the stir bar
spinning, and transfer the slides to this dish. Immediately add 625 μl
acetic anhydride, cover and wait 10 mins.
6) Wash 3 x 5 min PBS.
III. Pre-hybridization and Hybridization.
1) Set up a sufficient number of slide boxes to place all slides face up.
Cover bottom of boxes with a thick layer of paper towels and soak with 5xSSC,
50% formamide, about 50 ml or more per box. (2 boxes = 20 slides)
2) Place and cover slides with 200 μl hyb. sol. one at a time into the
humidifed box, without allowing the sections to dry. Seal chamber, let pre-hyb 2
or more hours at RT, checking periodically to be sure the slides haven't dried.
Hybridization 50% formamide 25 ml (de-I) 5 ml
Solution: 5X SSC 12.5 ml (20X, pH 7) 2.5 ml
5X Denhardts 5 ml (50X) 1 ml
250μg/ml baker's yeast RNA 1.25 ml (10 mg/ml) 125 μl 20mg/ml
500 μg/ml herring sperm DNA 2.5 ml (10 mg/ml) 500 μl
D E PC'd H20 3 . 75 m l 8 7 5 μl
total 50 ml 10 ml
3) Replace the pre-hyb with probe-containing hybridization solution
perpared as follows:
200 ng DIG-RNA / ml hybridization solution
Heat up to 80°C for 5 min, then chill on ice.
4) Place 75 μl of this solution per slide, and apply a coverslip (do each slide
one by one, no bubbles).
5) Incubate in the humidified slide boxes 68-70°C, O/N.
IV. Wash/Immunodetection.
1) After hybridization, slide off coverslips and place slides in 0.2X SCC,
72°C for 1 hour.
2) Move to RT and place dish containing slides in a new dish of 0.2X SSC
for 5 minutes.
3) Wash slides in buffer B1 (0.1M maleic acid, 0.15M NaCl, pH 7.5) for 5
minutes.
11.6 g Maleic acid
8.7 g NaCl
pH to 7.5 in total volume 1L.
4) Prepare Blocking solution (500μl 10% blocking soln. in 4.5 ml B1), 5ml
will cover 20 slides. Use 200μl per slide, in humidifed chamber, 1 hour.
5) Incubate with 1:2000 anti-DIG Ab in buffer B1 for 1 hour (still on
individual slides)
6) Rinse with buffer B1 30 mins, twice (transfer slides to dish)
7) Equilibrate with NTM (0.1M Tris pH 9.5, 0.1M NaCl, 5mM MgCl2)
10 ml 5M NaCl
25 ml 2M Tris, pH 9.5)
2 5 ml 1M MgCl 2
500 ml total
8) Color developing: in 200 ml NTM, add 880μl NBT, 660μl BCIP, and
0.048 g Levamisole, and mix. Add slides, cover with foil for desired period of
time.
9) Stop the reaction by transferring slides to PBS.