Protocol for In situ Hybridization on Paraffin section by DIG-Labeled RNA Probes
All the Procedures before hybridization should be done under RNAase-Free Conditions
All material that if possible should be autoclaved 2X.
Rat prostate tissues was dissected out in ice-cold PBS and fixed in 4% PFA overnight,
embedded in paraffin, and sectioned at 4uM. The slides are stored at room temperature.
Read this protocol all the way through before starting. Get all solutions and containers
ready!!
Steps for In situ:
 Incubate selected slides in 70 degree oven for 15min
 Place selected slides into autoclaved rack
 Dip into Xylene -------------------15min (2X)
 100% ETOH------------------------5min (2X)
 95% ETOH-------------------------5min
 70% ETOH -------------------------5min
 RNAse free H20--------------------5min
 RNAase free 1X PBS------------------5min
 4% PFA------------------------------10min
 RNAase free PBS-------------------5min 3X
 10ug/ml Proteinase K ( In 0.1M Tris-HCl ph 7.5 and 0.05M EDTA) 37°C for 30mins
(make 50ml for each jar in a 50ml polypropylene tube, pre-warm the buffer at 37° C for
10min, right before use add 10mg/ml Proteinase K 50ul, mix and put in jar with slides)
 At this point: Turn on Hybridization oven, preheat to 57°C
 1X PBS ------------------------------------5 min X 3
 0.1M Triethanolamine-----------------5min
 0.25% Acetic Anydride/0.1M Triethanolamine--10min (Make 50ml for each jar in a
50ml polypropylene tube, put 0.1M Triethanolamine 50 ml in the tube, right before use
add 125ul Acetic Anhydrate quickly, mix vigorously and quickly put into the jar with
slides)
 4X SSC briefly  2min
 Prehybridization-57°C for I hr (Use hybridization solution WITHOUT probe: either
place 50ul of hyb solution on each tissue, or soak in hyb solution)
 15min before hybridization
Denature 5ul sperm DNA, 10mg/ml Sigma, at 95°C for 5mins
o Cool on ice for 30 seconds
o Place in 500ul of Hyb solution
o Place 2ul-5ul of probe from in vitro transcription into 50ul hyb solution
o Mix well, denature at 75°C for 15mins
o Cool for 15min
o Use 20ul for each tissue
o Cover with hybrid-slip
 Hybridization overnight at 57°C ( Place a few drops of Water into hybridization chamber
After Hybridization: turn off Hybridization machine.

5X SSC
 60°C 10 min (Remove Hybri-slip)
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50% Formamide plus 2X - 60°C 30 min
2X SSC
 60°C 30 min
0.2X SSC
 60°C 30 min X2
1X Malaiec Buffer
 5 min
1X Blocking Buffer
 30 min
Sheep anti-DIG Fab Ab (1:1000 in 1X Blocking Reagent) incubate at 37°C for 2hr
1X Malaiec Buffer
15min X 2
Buffer II (Detection Buffer)
 5 min
Pipette out 1ml Buffer II, add 4.5ul NBT, 3.5ul BCIP, mix well, place immediately on
slides
Develop in a closed box for 30min-24hrs
Observe under microscope
After developing, place slides in Buffer II overnight
Wash with Water for 1hr
Dehydrate in ETOH, 50%, 75%, 100% ( For 5 seconds each)
Place in Xylene X 3 ( Dip and remove)
Mount with Permount or other mounting adhesive.
Take pictures of produced slides and store at room temperature.
Recipes for Solutions: All material must be RNAase Free!!! Autoclave whenever possible!
Hybridization Solution:
Final Concentration
50% Formamide
10% Dextran Sulfate
1X Danharts solution
10mM Tris-HCL ph7.5
60mM NaCl
1mM EDTA
0.25% SDS
1mg/ml tRNA
Stock Solution
100% Formamide
Dextran Sulfate Powder
50x Danharts solution
2M Tris-HCL ph 7.5
5M NaCl
0.5M EDTA
10% SDS
250mg/ml tRNA
Triethanolamine 7.6ml into 492.54ml RNAase free H20
Proteinase K Buffer:
0.1 M Tris-HCl ph 7.5 + 50mM EDTA
20X SSC
17.53g NaCl
8.82 Sodium Citrate
Dissolved in 80ml distill RNAase free Water, ph 7
Autoclave
50ml
25ml
5g
1ml
0.25ml
6ml
0.1ml
1.25ml
0.2ml
25ml
12.5ml
2.5g
0.5ml
0.125ml
3ml
0.05ml
0.625ml
0.1ml
10X Maleic Acid Buffer (MAB)
116g Maleic Acid
88g NaCl
Plus 800ml distill water, ph 7.5 with solid NaOH
QS to IL with water
Blocking Solution (provided)
Blocking Reagent (Roche) is dissolved in 1X MAB to a final concentration of 10% with shaking
and heating either on heating block or in a microwave oven.
Stock in aliquots at -20°C or at 4°C
Dilute with 1X MAB to 1% when using
1X Buffer II (Detection Buffer provided 10X)
1ml NaCl 5M
2ml MgCl 1M
5ml 1M, ph 9.5 Tris-HCL
24mg Levamisol final concentration of 2mM
QS to 50ml with distill autoclaved RNAase free Water
Protocol Adopted from Dr. Prins’ Lab UIC COM
Typed out by:
Obi Ekwenna
Dr. Hales’ Lab
UIC COM