The overall goal of this Micro RNA In Situ Hybridization is to detect the localization of the Micro RNA of interest in breast cancer tissue. So the message can answer the key question in breast cancer field. Such as the was the micro RNA of interest is unclear or tumor suppressant.
The major advantage of this technique is that it can detect cellular nuclearization of of interest in breast cancer tissue. Begin tissue preparation by removing paraffin from previously prepared breast tissue sections. Immerse the slides into a Coplin jar in fresh Xyline two times for ten minutes each.
Re-hydrate the tissue section with incubations in decreasing concentrations of ethanol in double distilled water for five minutes each. Next, immerse the tissue in diethylpyrocarbonate or DEPC treated water for five minutes. While the tissue is immersed make boundaries around the tissue section with a hydrophobic barrier pen.
After the DEPC immersion, fix the tissue with 4%PFA for 15 minutes. Follow the fixation with a PBS wash. Then, immerse the tissue in 0.3%Triton X-100 in PBS for ten minutes.
Incubate the sample with 50 micrograms per microliter of Proteinase K solution for 15 minutes at 37 degrees Celsius. Then incubate the tissue sections with triethanolamine and acetic anhydride for five minutes at room temperature. And follow with a PBS wash.
Wash the tissue thoroughly. And incubate the tissue section with 120 microliters of hybridization buffer for two hours at room temperature. Next, dilute the five prime DIG labeled NLA probe to a final concentration of 3 picamolar in 120 microliters of hybridization solution.
Then add three microliters of 40 nanograms per microliter stock to the 120 microliters of hybridization buffer. And incubate the mixture at 95 degrees Celsius for five minutes. Insert the tissue section into a ten by eight inch container and cover the tissue with 120 microliters of the probe so that it is completely covered.
Wet two paper towels and cover the container to create a humidity chamber. Incubate the tissue section overnight at 54 degrees Celsius. After hybridization, wash the tissue with five X saline sodium citrate buffer or SSC for seven minutes at 57 degrees Celsius.
Next, wash the tissue section with one X SSC twice. And wash the tissue section with 0.2 X SSC. Then, wash the tissue section with 0.2 X SSC for seven minutes at room temperature.
Incubate the tissue section in PBS for ten minutes. Incubate the tissue section with blocking buffer at room temperature for one hour in a humid chamber. Dilute the anti DIG AP antibody one to 300 in blocking buffer.
And incubate the tissue section with the antibody at 4 degrees Celsius overnight. The next day, wash the sections with wash buffer three times for five minutes each. Follow the ELF 97 commercial kit instructions to develop the tissue section with alkaline phosphatase that produces size three fluorescent light.
Next, wash the section with wash buffer for five minutes. Stain the section with 100 microliters of 2.5 nanograms per microliter Hoeshct dye for five minutes. And follow the stain with a five minute wash with wash buffer.
Use 15 microliters of mounting buffer per tissue section. And wipe excessive mounting solution with a tissue wipe. Then, seal the slide with clear nail polish to prevent leaking.
Finally observe the section with a fluorescent microscope at a total magnification of 100 X.Human breast cancer tissue from two patients was used to determine Micro RNA 489 expression. Memory gland duct and epithelial cells were found to express significantly higher Micro RNA 489 levels than adjacent tumor tissue. After watching this video, you should have a good understanding of how to perform Micro RNA In Sito hybridization by using breast cancer patient samples.
