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 -------------------à15min (2X)
• 100% ETOH------------------------à5min (2X)
• 95% ETOH-------------------------à5min
• 70% ETOH -------------------------à5min
• RNAse free H20--------------------à5min
• RNAase free 1X PBS------------------à5min
• 4% PFA------------------------------à10min
• RNAase free PBS-------------------à5min 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 ------------------------------------à5 min X 3
• 0.1M Triethanolamine-----------------à5min
• 0.25% Acetic Anydride/0.1M Triethanolamine--à10min (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 à 2min
• Prehybridization-à57°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

• Cool on ice for 30 seconds
• Place in 500ul of Hyb solution
• Place 2ul-5ul of probe from in vitro transcription into 50ul hyb solution
• Mix well, denature at 75°C for 15mins
• Cool for 15min
• Use 20ul for each tissue
• 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 à 60°C 10 min (Remove Hybri-slip)
• 50% Formamide plus 2X -à 60°C 30 min
• 2X SSC à 60°C 30 min
• 0.2X SSC à 60°C 30 min X2
• 1X Malaiec Buffer à 5 min
• 1X Blocking Buffer à 30 min
• Sheep anti-DIG Fab Ab (1:1000 in 1X Blocking Reagent) incubate at 37°C for 2hr
• 1X Malaiec Buffer à15min X 2
• Buffer II (Detection Buffer) à 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	Stock Solution	50ml	25ml
50% Formamide	100% Formamide	25ml	12.5ml
10% Dextran Sulfate	Dextran Sulfate Powder	5g	2.5g
1X Danharts solution	50x Danharts solution	1ml	0.5ml
10mM Tris-HCL ph7.5	2M Tris-HCL ph 7.5	0.25ml	0.125ml
60mM NaCl	5M NaCl	6ml	3ml
1mM EDTA	0.5M EDTA	0.1ml	0.05ml
0.25% SDS	10% SDS	1.25ml	0.625ml
1mg/ml tRNA	250mg/ml tRNA	0.2ml	0.1ml

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

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