NovaFISH is the first multiplex single-molecule fluorescence in situ hybridization (smFISH) probe developed by PixelBiosciences GmbH. NovaFISH can be used for detecting DNA/RNA expression and achieving digital quantitative analysis, suitable for isolated DNA/RNA, fixed cells, fixed tissue sections, or fixed whole embryo samples.

This manual is intended for the application of NovaFISH in fixed tissue samples, including tissue section preparation and NovaFISH staining steps.

Tissue Section Preparation

1. Tissue Block Preparation

For frozen tissue samples, quickly freeze the tissue sample using O.C.T. on dry ice (-78°C, within 3 minutes), or directly freeze in liquid nitrogen-balanced 2-methylbutane (approximately -150°C, within 1 minute). Store the tissue block at -80°C prior to sectioning. For FFPE (formalin-fixed paraffin-embedded) samples, follow standard procedures for formalin fixation and paraffin embedding. Paraffin-embedded tissue blocks can be stored at -20°C or -80°C.

Tip: NovaFISH is suitable for both frozen and FFPE tissue sections. To ensure RNA quality, tissue blocks should be stored at 4°C or lower to prevent chemical degradation and RNase-mediated degradation.

2. Tissue Sectioning and Mounting

Cut tissue blocks to a thickness of 3-5 μm for optimal NovaFISH staining. Mount the tissue sections onto 13 mm coverslips coated with poly-L-lysine or other amine-based coatings. For FFPE sections, bake the samples on a 37°C heating plate for an additional 2 hours to enhance adhesion. For frozen sections, section the tissue at -20°C using a cryostat (e.g., Leica Cryostat).

Tip:

• NovaFISH can also be used for thicker tissues (e.g., 10-20 μm) or even whole embryos of Platynereis (approximately 100 μm thick). Thinner sections generally reduce background noise and minimize diffraction artifacts during microscopy imaging.
• Frozen sections should be left at room temperature to ensure efficient binding to the coverslip. Once sectioned, immediately transfer the coverslip to a 24-well plate with dry ice, or store it at -20°C, then transfer the plate to a dry ice container, and ultimately store at -80°C.
• For the transportation of unfixed frozen sections, seal the entire 24-well plate (e.g., using tape) and place it in a container with sufficient dry ice to maintain low temperatures during transport. FFPE sections can be transported at room temperature.

3a. Fixation (Optional for Frozen Sections)

Place unfixed frozen sections in 4% formaldehyde (in 1xPBS solution) for 10 minutes. Then react with 135 mM glycine (in 1xPBS solution) for 10 minutes to quench the fixation reaction. Wash with 1xPBS for 10 minutes to remove residual formaldehyde and glycine. Finally, permeabilize the sample in -70% ethanol at 4°C overnight.

Tip:

• All buffers should be DEPC-treated solutions.
• After fixation, RNA stability is enhanced in frozen sections, making them somewhat resistant to RNase, but still susceptible to chemical degradation due to heat. Fixed sections should be stored in -20°C 70% ethanol, where RNA stability can be maintained for months to up to a year.
• Fixed samples are suitable for transport. Ensure they are kept at -20°C during transport, with sufficient 70% or higher ethanol concentration. Seal the 24-well plate during transport.

3b. Dewaxing (Optional for FFPE Sections)

Treat the paraffin sections on the coverslip with xylene twice, for 1 hour each. Then, wash off residual xylene with a gradient of 100%, 95%, and 70% ethanol, each step for 10 minutes.

Tip: Extending xylene treatment time (total of 2 hours) can maximize paraffin removal, thereby reducing background staining. Prolonged treatment times may damage the sample. RNA repair steps (e.g., heating in 0.01 M sodium acetate pH 6.0 at 80°C for 30 minutes) usually improve signal quality.

NovaFISH Staining

1. Probe Preparation

Resuspend the NovaFISH nano probe in 10 μL of DNase/RNase-free water (such as DEPC-treated water). For NovaFISH mini, midi, and maxi probes, resuspend in 40 μL, 200 μL, and 500 μL of water, respectively.

2. NovaWash Washing

Before hybridization, wash the coverslip twice at room temperature with NovaWash for 10 minutes each.

Tip: Using a shaker during washing may help remove residual ethanol more effectively.

3. NovaFISH Probe Staining

Add 0.5 μL of NovaFISH probe to 50 μL of NovaHyb solution to prepare the working solution. Draw a circle with glue on a clean glass slide to define the area for the solution, and add 10 μL of the working solution inside the circle. Place the coverslip with the cell side facing down onto the droplet. Seal the edges of the coverslip with glue. Keep a humid environment and incubate at 30°C overnight.

Tip: You can distinguish the cell culture surface from the non-culture surface by observing the coverslip. The cell culture surface typically has weaker reflection under light. Additionally, you can gently scratch the coverslip under the microscope—if cells fall off, it is the culture side.

4. Removal of Unbound Probes

Wash the coverslip four times at room temperature with NovaWash for 10 minutes each.

Tip: Alternatively, you can wash twice for 30 minutes each or wash overnight at 4°C, and the signal will still be preserved well. A contrast stain (e.g., DAPI) can be added during the first wash, with any excess stain removed during subsequent steps.

5. Mounting

Gently blot away any residual liquid from the coverslip using a clean tissue. Add 10 μL of mounting solution (Prolong Glass, ThermoFisher Scientific, Catalog Number: P36982) onto a clean glass slide and immediately place the coverslip with the cell side facing down. Store the sample at 4°C until use.

Tip:

• The sample can also be mounted directly with 2xSSC and imaged immediately.
• Imaging suggestion: Use an appropriate laser to image the sample with an epi-fluorescence microscope or confocal microscope (newer models usually provide better imaging quality). NovaFISH probes are labeled with combinations of Atto488, Atto565, and Atto647N.