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HYBRIDIZATION
1. In general, you will need to use 5 X.105 cpm of probe / slide, and 12 ul hybridization mix / 18 x 18 mm3 coverslip. This usually gives you optimal signal and low background, but for each new DNA probe you should experiment with different concentrations of probe per slide.
2. Calculate how much total probe needed making allowances f or about 33% more hybridization mix than needed for the number of slides used, and dry down this amount of probe (ex. for 10 slides, make enough hyb mix for 13 slides and calculate enough probe for 13, not 10 slides.)
3. Make stocks of solution A ( can store in freezer indefinitely in 1 ml aliquots)
" g dextran sulfate
10 ml 100% formamide
Vortex and heat to dissolve (50-650C)
4. Make up solution B as follows (ex: for 500ul hybridization mix)
hybrid mix
final conc Vol
5M NaCl 3OOmM 30 ul
2M TRIS 5OmM 12.5ul
0.15M EDTA imm 3.3 ul
100x Denhardts lx 5 ul
1M DTT lOmN 5 ul
BSA RNAase free 500ug/ml 5 ul
(50 mg/ml) (BRL)
t RNA (50 mg/ml) 500ug/ml 50 ul
sss DNA (2.5 mg/ml) 500ug/ml 100 ul
H20 + probe ---- 8% of total
volume
5. Add sufficient H20 to dessicated probe to equal 8% of total hybridization volume.
6. Final hybridization mix: (an example using a total hybridization mix volume of 500ul)
250 ul solution A
210 ul solution B
40 ul probe + H20
7. Remove slides from prehybridization mix and lay horizontally on table. Heat hybridization mix to 850C x 5 minutes, quick cool on ice and place 12 ul hybridization mix per slide.(if using 18 x 18mm coverslip; use 18 ul per 24 X 40 mm coverslip) . Cover with silanized coverslip taking care not to have any bubble formation. Seal with rubber cement. Ylace slides in slide holder, seal in two seal-a-meal plastic bags. Incubate slides in 600C water bath overnight.
(Note: temperature for incubation depends on GC content of probe and whether RNA-RNA or RNA-DNA or both are to be detected).
WASHES
1. Remove slides from bath. Carefully remove rubber cement from coverslips and place slides (with coverslips still on) in 2 x SSC at RT on shaker for 5-10 minutes until coverslips begin to slide off. For those slides whose coverslips have not fallen off, remove carefully, trying not to remove tissue along with coverslip.
2. 50% Formamide, 2xSSC wash at RT x 15 minutes on shaker.
3. Three 2 x SSC, 0.1% SDS washes at 650C x 15 minutes in shaking water bath.
4. RNAass wash 370 Cx 30 minutes
2M TRIS 0.3M 0.75cc
5M NaCl 10 mm 9 cc
10 mg/ml RNAaseA 40ug/ml 600 ul
1.5 mg/ml RNAaseTl 5 ug/ml 500 ul
qs H20 qs 150 ml
5. 2 x SSC, 0.1% SDS at 65C x 15 minutes x 2
6. 0.1% SSC. 0.1% SDS at 65c x 15 minutes x 2
7. Dip 15 times in 70% ETOH x 2 Dip 15 times in 100% ETOH x 2
8. Air dry
AUTORADIOGRAPHY
1. Emulsion (NTB-2, Kodak) should be used no more than 2 months
before discarding. Diluted emulsion can also be reused, but again, only for about two months (which is usually shorter than the expiration date).
2. Take the bottle of emulsion, and in complete darkness, wrap heavily in aluminum foil and melt in 450C water bath in darkroom. In addition, heat equal volume (110 ml) d2H20 at 450C. While still in absolute darkness, dilute emulsion 1:1 with H20, mixing gently (or emulsion will autodevelop!.'), and aliquot back into parent bottle and other bottles or Falcon tubes. Cover all bottles containing emulsion with triple-layer foil so they are light tight. They can now be stored in refrigerator between uses.
3. Pour about 25 cc emulsion into coplin dish sitting in water bath. Dip a few blank slides to eliminate bubbles.
4. Set up slides in blocks of 5 in slide holders.
5. Dip slides in emulsion, counting to ten while withdrawing them
steadily from the emulsion. Blot on paper. Place in slide Dryer (Oncor) and dry-for 10-15 minutes (until emulsion dry). Then stack slides in light tight slide boxes with dessicant, wrap with foil and label.
6. Store in -700C freezer for duration of exposure (2 days to 2 weeks, depending on strength of probe and signal).
SLIDE DEVELOPMENT
1. Dissolve Kodak D-19 developer powder in 3.8L 500C water. Store in dark or covered bottle. Remake every 4 weeks.
2. Make up a solution of f ixer as follows (can be used within a week):
30% Nathiosulfate solution: 90 grams Na thiosulfate
(Sigma S-8503) in 300 cc dH20
3. IN ABSOLUTE DARKNESS (perform in ice bath) :
a. Dip slides in developer (300cc) for 4 minutes. Developer solution should be in ice bath at 150C during development, not more or less!!!
b. Dip slides in distilled water (300cc) to which is added 2 cc 100% acetic acid for 30 seconds.
c. Dip slides in fixer for 2 1/2 'minutes (300cc).
d. Dip slides in distilled water for 5 minutes, changing bath x 2.
STAINING OF SLIDES
1. Filter Hematoxylin stain over Whatman paper. Dip slides in Hematoxylin stain (from American Histolabs) for 10 minutes.
2. Destain in lukewarm H20 for 10 minutes until water clear and cells begin to blue under scope
3. Dip in 70% ETOH x 2
4. Dip slides in Eosin stain (American Histolabs) for 2 ½ minutes.
5. Pass through 70% x 2, 95% x 2, 100% x 2, xylene x 2.
6. Let air dry.
7. Place drop of Permount on slide, seal with coverslip.
PROBE CALCULATIONS:
i.cpm/ul (date)
2.#slides
3.#slides X 1.5
4.cpm/slide
5.total cpm needed (3 x 4)
6.total ul probe (5/1)
HYBRIDIZATION MIX CALCULATIONS:
slides
slides X 1.5
Hyb mix/slide
total hyb mix needed
GRAND TOTAL HYB MIX
ADD 50-75 ul TO GRAND TOTAL HYB MIX VOLUME AND ROUND OFF AMOUNT TO
NEXT HIGHEST MULTIPLE OF 100:
THEORETICAL HYB MIX VOLUME
SOLUTION B: TO CALCULATE, MULTIPLY VOLUME PER 100 ul HYB MIX BY
GRAND TOTAL HYB MIX VOLUME TO BE MADE, DIVIDED BY THE MULTIPLE OF
100 USED ABOVE
STOCK SOLUTION FINAL CONC VOLUME MULTIPLE TOTAL
PER 100 ul OF 100 VOLUME
5M NaCl 3OOmM 6 ul
2M TRIS 5OmM 2.5 ul
0.15M EDTA imm 0.7 ul
100x Denhardts lx 1 ul
1M DTT iomm 1 ul
BSA RNAase free 500ug/ml 1 ul
(50 mg/ml) (BRL)
t RNA (5 mg/ml) 500ug/ml 10 ul
sss DNA (2.5 mg/ml) 500ug/ml 20 ul
FINAL HYBRIDIZATION MIX: PROBES:
Solution A (50% of total)
Solution B (42% of total)
DEPC d2H20
Probe (8% of total)
DAY TWO: WASHING
1. 2x SSC, RT, 15 min x 1
2. 50% formamide, 2x SSC, RT, 15 min x 1
3. 2x SSC, 0.1% SDS, 650C, 15 min x 3
4. RNAase wash, 370C, 30 min x 1 (prewarm prior to use):
2M TRIS 0.3M 0.75cc
5M NaCl 10 mm 9 cc
10 mg/ml RNAaseA 40ug/ml 600 ul
1.1 mg/ml RNAaseTl 5 ug/ml 680 ul
qs H20 qs 150 ml
5. 2x SSC, 0.1% SDS, 650C,15 min x 2
6. O.lx SSC, 0.1%SDS, 650C, 15 min x 2
7. 70% ETOH, 15 dips
8. 100% ETOH, 15 dips
9. Let air dry
10. Dip in emulsion.
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