From Jimmie Fata 2/18/03
In Situ Hybridization Method on Mammary Sections Using DIG Probes
1. Dewax and Rehydrate paraffin embedded sections
a. 2 X 2 minutes toluene
b. 2 min 100% ETOH
c. 2 min in 95% ETOH
d. 2 min in 80% ETOH
e. 2 min in 70% ETOH
f. 2 X 2 min in 2X SSC buffer
2. Proteinase K Digestion
a. Pro K treatment @ RT for 10 min
b. Wash Slides 2 X 5 min in 2X SSC buffer
3. Acetic Anhydride Treatment
a. Acetic Anhydride treatment @ RT for 10 min (add A.A. just before adding slides to the wheaton jar and put stir bar at bottom)
b. Wash Slides 2 X 5 min in 2X SSC buffer
a. Use a wax pen to delineate mammary gland on section
b. Prehybridize in prewarmed Pre-Hyb. Buffer (100ml) for 2-4 hours @ 50 degrees C in a humidified chamber that has paper towels soaked in 50% formamide in 5X SSPE Buffer.
a. Remove excess prehyb. Buffer w/kimwipe, avoid contacting tissue.
b. Add probe (100 ng) directly to Pre-hyb. Buffer and (400 ng) tRNA.
c. Hybridize laying flat in a humidified chamber
d. Incubate @ 50 degrees 12-36 Hours
a. Wash sections 4X in 4X SSC 10 minutes each
7. DIG Primary Antibody
a. After last wash, rinse 5 min in Buffer 1
b. Transfer to blocking buffer for 1hr @ RT
c. Wipe slide with kimwipe, use wax pen to outline tissue then add 100ml antibody in blocking buffer, incubate for 4 hours @ RT in a light-sealed chamber with paper towels moistened in buffer 1
a. Wash 3 X 10 min in buffer 1
b. Wash 5 min in buffer 2 for equilibriating
9. DIG Probe Color Detection
a. After last wash, wipe slide with kimwipe, add 450 ml color development solution.
b. Incubate slide with C.D.S. for X hours @ RT in a sealed container lined with paper towels moistened in buffer 2.
c. Stop Reaction by transferring slides to buffer 3 (To check the progress of the staining you can temporarily stop the reaction in buffer 2 then add more CDS).
10. Staining Nucleiand Photography
a. Dip 4X in dH20.
b. Stain in nuclear fast red (.1mg/ml in water) 2 min
c. Destain briefly in dH20.
d. Pass through graded alcohols 70% to 100% (2 min).
e. Dip slides in Xylene
f. Coverslip with permount.
Proteinase K treatment: (prepare fresh-20mg/ml pro K in 20mM Tris-Cl pH 7.5, 2mMCaCl)
20X SSC (1 Liter): (175.3g NaCl, 88.2g Na Citrate in DEPC treated H20), prepare 2X about 2 liters, and 4X about
20X SSPE (1 Liter): (175.3g NaCl, 27.6g NaH2P04.H20, 7.4g EDTA in 800mls, adjust pH to 7.4 with NaOH ( about 6.5 mls 10N NaOH), autoclave.
Acetic Anhydride Treatment: (prepare fresh, 250 mls of .1M triethanolamine+1.25 mls acetic anhydride mixed on stir plate in hood, mix 30-60 seconds until all emulsion beads are gone)
Prehybridization Buffer: (50% formamide, 5X SSPE, 1X Denhardts Solution)
Hybridization Probe: (20ml prehyb, 100 ng DIG labeled riboprobe, and 400 ng tRNA)
Buffer 1 (1.5 Liters): (100 mM Tris-Cl pH 7.5, 150 mM NaCl)
Blocking Buffer (30 mls): Buffer 1 + .3% triton X-100, 2% normal sheep’s serum)
Antibody Solution: (1:500 dilution goat anti-DIG antibody in blocking buffer)
Buffer 2 (50 mls): (100 mMTris-Cl pH 9.5, 100 mM NaCl, 50 mM MgCl2)-for equilibriating
Color Development Solution: (10 mls buffer 2, 35 ml 100 mg/ml NBT, 35 ml 50 mg/ml BCIP, 25 ml 1 M livamisol- to block endogenous peroxidase activity-60mg in 250 ml water = 1M)
Buffer 3 (30 mls): (20 mM Tris-Cl, 10mM EDTA)
Nuclear Fast Red: (.1 mg/ml in water)
DIG labeling mix (Boeheringer Mannheim)
5-bromo-4-chloro-3-indol-phospate (BCIP 150 mgs in 3 mls 70% DMF)
4-nitro blue tetrazolium chloride (NBT 300 mgs in 3 mls 70% DMF)
normal sheep’s serum (Gibco)
 All glassware should be washed with Absolve glassware cleaner (NEN-Dupont) prior to the protocol to remove RNASE.
 For frozen sections start here.
 Opens up tissue to allow probe access, a timecourse experiment was performed on paraffin embedded embryos and little signal intensity was seen between 5-10 minutes of digestion. After 20 mins the tissue began to fall off slide, no signal.
 Neutralizes positive charges on protein in the tissue, prevents charge interactions with probe.
 Blocks non-specific binding-Upright slide (Coplin Jars) work well 8-12 slides per 30 mls prehyb.
 TRNA is acts as a carrier for RNA.
 Prehyb and Hyb can be performed without coverslips. Dry slide w/kimwipe, circle tissue with was pen, add 100 ml prehyb and place in a sealed box humidified with paper towels soaked in 50% formamide/5X SSPE. After 2-4 hours at 50 degrees Celsius, add probe directly to prehyb and incubate at 50 degrees overnight.
 X hours can equal 30 minutes for very abundant mRNAs like U2 or up to 24 hours for less abundant ones like MMP-9. Most reasonably abundant mRNAs can be detected overnight (12-18 hours). Can stop reaction in buffer 2 to look at reaction, then just add more CDS. Stop reaction in buffer 3. For longer incubation times re-adding CDS is recommended. Background will increase with time therefore one must optimize incubation times for best signal to noise ratio.
 If background staining is light you can stain nuclei using this stain.