REAGENT SETUP
LNA DIG labelled probes:
Probes were purchased labelled with digoxigenin at 5’ and 3’ (25 µM). Probes were diluted and stored according to published protocol 18 and manufactures instruction. Pre-hybridizations and hybridizations were performed at temperature 20-25°C below the Tm of the given LNA probe. Working conditions and the efficiency of the in situ hybridizations were determined by using both positive (U6, miRNA known to be ubiquitously expressed in most of the tissues) and negative (scramble, has no homology to any known miRNA/ mRNA sequence) control probes
Hybridization Mix 10ml:
According to published protocol 18 (To 1.8 ml DEPC-treated water add 5ml of ultra pure 50% formamide, 2.5ml of 20x SSC, 500μl of 10 ug/μl yeast t-RNA, 200 μl of 50x Denhardt’s solution for a total volume of 10ml. Divide into aliquots and keep at -20°C. The hybridization buffer has a pH of 6-6.5. If the hybridization temperature exceeds 55°C; add citric acid to 9.2mM final concentration).
Proteinase K buffer: 100 mM Tris-HCL, pH 8 and 50 mM EDTA, pH 8.
AP buffer: 100 mM Tris HCl pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20
DEPEC treated water: Add 1 ml of DEPC to 1L water, mix it well and leave overnight in a fume hood. Next day autoclave the solution.
EQUIPMENT SETUP
Humidified slide incubation box:
We used metallic box (28.5X18.5 cm) for incubating the slides for pre-hybridization and hybridization. Piece of sponge of same size as that of box was kept inside where 5XSSC was poured to maintain the moist conditions.
PROCEDURE
Preparation of paraffin tissue sections
1) Cut 5 µm paraffin sections from paraffin block and place them at 37°C incubator overnight for drying.
CRITICAL STEP: Sections once cut can be stored at room temperature and can be used within 1 week. We have not tested the in situ signals from samples older than 1 week.
Deparaffinization, deproteination, pre hybridization, hybridization and washes were done according to Jorgensen et al with some modification.
Deparaffinization
2) Treat the paraffin sections slides 3 times 5 minutes with xylene.
3) Immerse the slides in 100% ethanol 2 times for 5 minutes.
4) Immerse the slides in 70% ethanol for 5 minutes.
5) Immerse the slides in 50% ethanol for 5 minutes.
6) Immerse the slides in 25% ethanol for 5 minutes.
7) Wash the slides with DEPC water for 1 minute.
Deproteination
8) Wash the slides 2 times 5 minutes in PBS.
9) Treat the slides for 5 minutes with proteinase K (10 µg/ml) at RT.
CRITICAL STEP: The signals obtained using proteinase K treatment according to published protocols at best gave weak signals that were difficult to interpret. Different concentration and temperature for proteinase K treatment were tested for different tissues like serous ovarian carcinoma, lymph nodes, endrometrium, fallopian tube, small intestine, breast and ovarian cancer; 10 µg/ml of proteinase K at RT gave the best results for all the tested tissues.
10) Stop the proteinase K reaction by treating the slides for 30 second in 0.2% glycine in PBS.
11) Rinse the slides 2 times in PBS.
12) Wash the slides 2 times 5 minutes in PBS.
Pre-hybridization
13) Pre-hybridize the slides in hybridization buffer according to published protocol18 for 1 hour at the probes hybridization temperature.
CRITICAL STEP: Pre-hybridization and hybridizations conditions for the probes should be 20-25°C below the Tm of the probe as recommended by the manufacturer (www.exiqon.com).
Hybridization
14) Mix 2.5 pmoles of diluted probe in 100-200 µl of hybridization buffer. Probes were diluted according to published protocol 18.
15) Pipette the hybridization mix on the slide.
16) Place the slide in humidified incubation box for 2 hours.
Washes
18) Rinse the slides in water bath at hybridization temperature for 5 minutes in 2XSSC.
19) Wash the slides 3 times for 30 minutes in 50% formamide, and 2XSSC at hybridization temperature in water bath with shaker.
20) Wash the slides 5 times for 5 minutes in PBS at room temperature.
Chromogenic detection
21) Block the slides at room temperature for 1 hour in blocking buffer (2% sheep serum in PBST).
22) Incubate the slides overnight with antibody (1:2000) at +4 °C.
23) Wash the slides for 3-4 times in PBST.
24) Wash the slides 3-4 times in AP buffer
25) Cover the slides with BCIP/ NBT liquid substrate in a humidified chamber at until the colour develops
26) Stop the colour reaction by washing the slides with PBST for 15 min.
27) Dry the slides, (do not over dry) and mount the slides gently using aquamount mounting medium.
28) Image the slide using any suitable conventional microscope.