Choose an appropriate RNA probe template.
RNA probes should be between 250 to 1500 nt in length. Probes approximately 800 nt long exhibit the highest sensitivity and specificity. Ideally transcription templates should allow for transcription of both probe (antisense strand) and negative control (sense strand) RNAs. Cloning into a vector with opposable promoters will achieve this. Remember that circular templates must be linearized before making a probe. PCR templates can also be used.

Modify protocol if using DNA probes.
DNA probes can provide equivalent sensitivity to RNA probes. However, DNA probes do not bind as tightly to the target mRNA molecules in the tissue sections. Therefore, formaldehyde should not be used in the post hybridization washes when using DNA probes.

Use an appropriate radiolabel.
Use ³⁵S-NTP to label probes for in situ hybridization of tissue microarrays. ³²P is a high ß particle emitter and creates "bleed" which interferes with analysis. While ³³P is O.K. for ISH experiments of many types of tissue sections, it can also cause "bleed" when used with the small tissue microarray specimens, and is therefore not recommended.

Change reagents frequently.
Triethanolamine and acetic anhydride should be changed out once every 2-3 weeks and 10% neutral buffered formalin (NBF) should be replenished once every 3-4 days. ³⁵S-labeled probes can be stored at ­20°C for up to 8 weeks after synthesis. Hybridization signal will decrease with time due to radioactive decay of the probe.

Optimize Proteinase K digestion.
Proteinase K digestion is a critical step for successful ISH. Insufficient digestion will result in a diminished hybridization signal. On the other hand, if the sample is over digested, tissue morphology will be poor or completely destroyed, making localization of the hybridization signal impossible. The concentration of Proteinase K needed will vary depending upon the tissue type, length of fixation, and size of tissue core. In our experience with a wide variety of tissues, 1-5 µg/ml Proteinase K for 10 min at room temperature works well. The best way to determine optimal Proteinase K digestion conditions is to do a Proteinase K titration experiment and then hybridize with a probe known to bind to RNA in the tissue. Choose the Proteinase K digestion conditions that produce the highest hybridization signal with the least disruption of tissue or cellular morphology.

Protect hybridizing slides.
Plastic coverslips or Parafilm^® should be used for tissue microarray hybridizations. Glass coverslips can create a vacuum that can pull the small tissue cores off the slide.

Optimize hybridization temperature.
Hybridization temperature depends upon the tissue type and should be optimized for each tissue microarray analyzed. Typical hybridization temperatures range between 55 and 62°C.

Use RNase-free reagents.
Make sure that all reagents and supplies that come in contact with the tissue microarray slides are RNase-free. Because of the RNase digestion step in the post hybridization processing, all glassware and slide holders used for post hybridization washes should be reserved only for post hybridization processing and separated from glassware used in earlier steps in the procedure.

Test new probe preparations and slides.
Test new probe preparations on sections that are known to contain an easily detected amount of target before using it with experimental slides. This is especially important when experimental slides contain sections that are expensive or difficult to obtain.

Avoid formation of air bubbles.
It is convenient to apply the probe solution to the coverslip, lay the section face down on top of the hybridization solution and invert the slide plus coverslip so that it is right side up. If air bubbles are trapped underneath the coverslip, remove them by gently pressing on the cover glass to move them to the side.

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