Materials and Reagents

Gloves
Tissue slides
Superfrost plus slides
Coverslips
LNA^TM Scramble-miR probe (Exiqon, catalog number: 699004-370 )
LNA^TM miR-409-3p 5’ biotin labeled (Exiqon, catalog number: 610701-370 )
LNA^TM miR-409-5p 5’ biotin labeled (Exiqon, catalog number: 615615-370 )
RNaseZap (Thermo Fisher Scientific, Ambion^TM)
Xylene
Ethanol
Sterile PBS (pH 7)
Nail polish
Horse serum (Vector Laboratories, catalog number: S-2000 )
Streptavidin block reagent (Thermo Fisher Scientific, Invitrogen^TM)
Streptavidin conjugated QDs 625 nm (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: A10196 ) (1 µm stock from Invitrogen)
Streptavidin conjugated QDs 565 nm (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: Q10131MP ) (1 µm stock from Invitrogen)
6% IgG-free, protease free BSA (Jackson ImmunoResearch, catalog number: 001-000-162 )
BSA
0.4% Triton X-100
0.1% Tween-20
Mounting media
4’6-diamidino-2-phenylindole (DAPI) (Vector Laboratories)
1 M Tris-HCl (pH 7.4)
0.5 M EDTA
NaCl
RNase free Milli-Q water, autoclaved (all solutions prepared in this water)
Exiqon microRNA ISH buffer set and Proteinase K (Exiqon, catalog number: 90000 )
20x SSC, pH 7.0
Proteinase K stock (see Recipes)
Proteinase K buffer (see Recipes)
Proteinase K solution (15 µg/ml) (see Recipes)
SSC solutions (see Recipes)
Hybridization mix (see Recipes)
Streptavidin blocking solution (see Recipes)
QD solutions (see Recipes)
PBS-T (see Recipes)

Equipment

Autoclave
Hybridizer (DAKO Statspin Hybridizer)
Glass cutter
Slide rack and glass jars
PAP pen or ImmEdge pen (Vector Laboratories, catalog number: H-4000 )
Water bath
CRi multi-spectral camera with built-in Nuance software and inForm software (PerkinElmer, Waltham, MA)

Software

Nuance v3.1 software
Inform v1.3 software
Graphpad Prism software

Procedure

Notes:

The in situ hybridization procedure has been described in detail in the Exiqon ISH protocol.
Caution of RNA work

All steps should take place in a clean and nuclease free environment.
All surfaces should be cleaned with RNaseZap.
Wear gloves at all times and use autoclaved glassware and RNase grade MilliQ water which is autoclaved for preparation of all solutions.
In situ hybridization was performed as mentioned in the Exiqon manual.

Optimization

Optimization of Proteinase K treatment required before the start of the actual assay. Proteinase K reagent prepared immediately before use at a concentration of 15 µg/ml.
Optimization of LNA miRNA probe and scramble miRNA. A probe concentration of 80 nM was optimal for detection of miR-409-3p and miR-409-5p.

Reagents to be prepared on the day of the experiment

Proteinase K
Hybridization mix: Place the appropriate amount of probe in a 2 ml non-stick RNase-free tube. Denature the probe at 90 °C for 4 min. Centrifuge the tubes and spin down. Add 2 ml of 1x miRNA ISH buffer.

Deparaffinize slides in xylene and ethanol
1. Xylene - 5 min, 3 times in a glass jar
2. 99.9% ethanol - 10 min, 2 times, in a glass jar
3. 99.9% ethanol - 5 min, 1 time, in a glass jar
4. 96% ethanol - 10 min, 1 time, in a glass jar
5. 96% ethanol - 5 min, 1 time, in a glass jar
6. 70% ethanol - 10 min, 1 time, in a glass jar
7. 70% ethanol - 5 min, 1 time, in a glass jar
8. PBS - 2-5 min, each 1 time, in a glass jar
Proteinase K treatment and wash
1. Add Proteinase K to Proteinase K buffer (final concentration 15 µg/ml) (add 7.5 µl Proteinase K stock to 10 ml Proteinase K buffer).
2. Add sufficient volume of proteinase K solution to cover the slides (instructions in the Exiqon manual).
3. Incubate for 10 min, at 37 °C, in a Hybridizer.
4. Transfer the slides into a new glass jar containing PBS. Repeat this step twice.
Hybridization
1. Use PAP pen to mark around the tissue section.
2. Using a glass cutter cut the coverslip (autoclaved) to the size of the tumor tissue section.
3. Add miRNA detection probe (50 µl, hybridization mix) on the tissue section. Avoid air bubbles.
4. If a slide has several serial tissues sections of the same tumor, it can be used to determine several probes (such as miR-409-3p, miR-409-5p and Scramble miRNA). Once the probe is added, place the cut coverslip very carefully without trapping air bubbles.
5. Seal the edge of the coverslip to the slide using colorless nail polish. Let it dry.
6. Place the slides in the Hybridizer at 55 °C for 1 h.
Disassembly and washes
1. Carefully remove the coverslips.
2. Place the slides into a glass jar with 5x SSC in a water bath at 55 °C.
  Note: Use a water bath at 55 °C to do the washes in glass jars. The graded SSC solutions were prepared with autoclaved water.
3. 5x SSC - 5 min 55 °C
4. 1x SSC - 5 min 55 °C
5. 1x SSC - 5 min 55 °C
6. 0.2x SSC - 5 min 55 °C
7. 0.2x SSC - 5 min 55 °C
8. 0.2x SSC - 5 min RT
9. Incubate the slides in PBS.
Blocking
The slides were incubated in PBS containing 2.5% horse serum and 20% streptavidin block reagent for 1 h at room temperature.
Single QD labeling
1. Streptavidin-conjugated QDs (565 nm [green] and 625 nm [red]) were prepared at a 10 nM concertation in PBS-T with 6% IgG-free, protease free BSA.
2. The slides were incubated at 37 °C for 60 min.
Washing
The slides were washed 3 times for 5 min each in PBS-T (PBS + 0.4% Triton X-100 + 0.1% Tween-20).
Mounting
Cover the slides with cover glass using aqueous mounting media containing 4’6-diamidino-2-phenylindole (DAPI).
Image acquisition
The CRi spectral imaging system was used. Multispectral images were taken using the Nuance v3.1 software. For each field of the tissue, serial images were taken from 450 to 800 nm with an interval of 10 nm. This generates a cube a stack of 36 separate images at different wavelengths and has spectral information for every pixel in the image. Images are acquired at 400x magnification (Figure 2).

Figure 2. Representative images of multiplexed image of miR-409-3p (green) and miR-409-5p (red) on a human prostate cancer tissue sample of Gleason grade 8, detected using in situ hybridization and quantum dots labeling. DAPI was stained in blue. Scrambled miRNA was used as a negative control, on a adjacent section on an separate slide of the same tissue. Hematoxylin and eosin staining (H&E) was also performed. (Magnification 40x)

Data analysis

Once data acquisition is performed the images are deconvoluted or unmixed to extract the specific signal by a specified QD. A spectral library was built for 565 and 625 nm. The spectral library was used to unmix the cube. Autofluorescence was reduced using the Real Component Analysis plug-in software. The images after unmixing represented the distribution of QDs on the tissue. Signal was quantified using Inform v1.3 software. The software discriminates cancer and stromal (non-cancer) areas. The software also discriminates between nuclear and cytoplasmic area based on the DAPI stain. Using this software the QD signal from the tumor and stromal sections can be determined and quantified. Numerical values are generated for the image and the cytoplasmic component of the tumor and stromal regions is used for plotting the data. The data is graphically plotted using Graphpad Prism software. Data distribution was depicted in box plot formats. Statistical analysis of tissue array was performed using a non-parametric Wilcoxon rank sum test. Values of P < 0.05 were considered to be statistically significant.

Notes

Multiplexed QD

Principle: Multiplexed ISH-QD labeling involves using a single tissue which is labeled with a miRNA-biotin probe using ISH and followed by a streptavidin blocking step and incubated with streptavidin conjugated-QD. This process is repeated with another miRNA-biotin probe and another (different wavelength) streptavidin conjugated-QD pair. Finally the tissue is stained with mounting media containing DAPI. Thus, an image is generated for different miRNA with different active fluorescent QD on a single tissue section.
Procedure: Slides were stained sequentially, with one miRNA probe and a QD procedure followed by another probe and QD. After the first QD labeling, slides were washed 3 times for 5 min each with PBS-T. Slides were then used for hybridization (Procedure C) with another miRNA probe and continued to Procedure H. Hybridization mix and probes stable for only 6 months, avoid multiple freeze-thaw cycles.

Freshly prepared tissue slides give better miRNA signals. Older tissue preparations, may have loss of miRNA content due to RNase degradation over time.

Recipes

Proteinase K stock
Reconstitute 20 mg/ml in 600 µl of 10 mM Tris-HCl, pH 7.4 (RNase-free). Store at -20 °C
Proteinase K buffer
5 ml of 1 M Tris-HCl (pH 7.4)
2 ml of 0.5 M EDTA
0.2 ml of 5 M NaCl
900 ml of RNase-free water
Adjust the volume to 1,000 ml and autoclave
Proteinase K solution (15 µg/ml)
Before the start of the experiment add 7.5 µl of Proteinase K stock to 10 ml Proteinase K buffer to make a concentration of 15 µg/ml
SSC solutions
Stock: 20x SSC solution
5x SSC: 1 L = 250 ml 20x SSC + 750 ml water
5x SSC: 1 L = 50 ml 20x SSC + 950 ml water
5x SSC: 1 L = 10 ml 20x SSC + 990 ml water
All solutions to be autoclaved
Hybridization mix (miRNA ISH buffer and LNA^TM detection probes)
Note: LNA^TM miRNA detection probes come in 40 µl volume at a 25 µM concentration.

Add 1 ml 2x ISH buffer to 1 ml of RNase free water
Denature the LNA^TM miRNA detection probes at 90 °C for 4 min before adding the ISH buffer. Spin down the solution
For making 80 nM of miRNA probes, add 6.4 µl of denatured probe (25 µM) to 2 ml of 1x ISH buffer
Note: Hybridization mix can be stored at -20 °C for six months.

Streptavidin blocking solution
PBS containing 2.5% horse serum and 20% streptavidin block reagent
QD solutions
Streptavidin-conjugated QDs (565 nm [green] and 625 nm [red]) was prepared at a 10 nM concertation in PBS with 6% IgG-free, protease free BSA
PBS-T (PBS + 0.4% Triton X-100 + 0.1% Tween-20)
Add 1 ml of Tween-20 and 4 ml of Triton X-100 to 1 L PBS
Autoclave at 121 °C for 15 min

Acknowledgments

This procedure has been published in part in the paper by Hu et al., 2011 and Josson et al., 2015. The in situ hybridization procedure has been optimized from the Exiqon ISH protocol. Grant support for this work is from P01-CA98912, DAMD-17-03-02-0033, RO1-CA122602 (L.W.K. Chung).

References

Hu, P., Chu, G. C., Zhu, G., Yang, H., Luthringer, D., Prins, G., Habib, F., Wang, Y., Wang, R., Chung, L. W. and Zhau, H. E. (2011). Multiplexed quantum dot labeling of activated c-Met signaling in castration-resistant human prostate cancer. PLoS One 6(12): e28670.
Josson, S., Gururajan, M., Sung, S. Y., Hu, P., Shao, C., Zhau, H. E., Liu, C., Lichterman, J., Duan, P., Li, Q., Rogatko, A., Posadas, E. M., Haga, C. L. and Chung, L. W. (2015). Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis. Oncogene 34(21): 2690-2699.