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.

NovaFISH can also be combined with immunofluorescence staining (IF) to simultaneously detect RNA (via NovaFISH) and protein (via immunofluorescence staining) in fixed cells. This manual includes the following experimental steps: Immunofluorescence Staining and NovaFISH Staining.

Immunofluorescence Staining

1. Cell Fixation

Culture cells on 13 mm #1 glass coverslips. Fix the cells at room temperature with 3% PFA/PBS for 10 minutes. Wash with 1 mL PBS at room temperature for 1 minute. Wash with 0.5% Triton-100/PBS at room temperature for 10 minutes, then wash with PBS at room temperature for 1 minute.

2. Primary Antibody Incubation

Add primary antibody and incubate at room temperature for 1 hour. Wash with PBS at room temperature 4 times, each for 5 minutes.

3. Secondary Antibody Staining

Add secondary antibody and incubate at room temperature in the dark for 1 hour. Wash with PBS at room temperature 3 times, each for 5 minutes.

Tip: NovaFISH staining should follow the immunofluorescence staining steps immediately to preserve the signal.

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.

Tip: Lightly tap the tube multiple times to help dissolve the probes or leave the tube at room temperature for 20 minutes. NovaFISH probes should be stored at -20°C or lower. Repeated freeze-thaw cycles will not affect product quality. All water used in the following steps should be DEPC-treated to minimize RNase contamination.

2. Cell Preparation

Fix the cells, which have completed antibody staining, at room temperature with 1x PBS solution containing 4% formaldehyde for 10 minutes. After removing the formaldehyde, wash with 1x PBS solution containing 135 mM Glycine for 10 minutes to remove residual formaldehyde.

Tip: If your cell line requires coating, use a suitable reagent for cell attachment and growth on the coverslip. Cells should grow overnight at varying densities (10%-100%) to ensure proper attachment before fixation with 4% formaldehyde. Fixation can also be done using 1x PBS solution containing 4% paraformaldehyde. It is recommended to use a 12-well or 24-well plate for cell culture, fixation, and washing steps.

3. Remove Residual Formaldehyde

Wash once with 1x PBS and incubate for 10 minutes to remove residues. Then, place the sample in 70% ethanol and store at 4°C overnight.

Tip: Cells can also be stored in 70% ethanol at -20°C for several months. It is recommended to seal the culture plate containing fixed cells with sealing film or tape to prevent ethanol evaporation. Regularly check the ethanol level in the wells to ensure the cells do not dry out.

4. NovaWash Washing of Cells

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

Tip: A shaker can be used for washing to better remove residual ethanol.

5. NovaFISH Probe Staining

Add 0.5 μL of NovaFISH probe to 50 μL of NovaHyb solution to prepare the working solution. Use glue to draw a circle on a clean glass slide to contain the solution. Add 10 μL of the working solution inside the circle. Place the coverslip (cell side down) onto the drop of solution. Seal the edges of the coverslip with glue. Maintain a humid environment and incubate at 30°C overnight.

Tip: The cell culture surface can be distinguished from the non-cultured surface by observing the reflection under light. Additionally, you can gently scratch the coverslip under a microscope, and if cells fall off, it indicates the cell side.

6. Remove Unbound Probes

Wash the coverslip in the culture well with NovaWash 4 times, 10 minutes each time, at room temperature.

Tip: You may choose to wash twice for 30 minutes each, or wash overnight at 4°C. The signal will still remain strong. You can add counterstains (such as DAPI) during the first wash and remove excess dye in subsequent steps.

7. Mounting

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

Tip: Use an appropriate laser for imaging the sample with an inverted fluorescence microscope or confocal microscope (newer models typically provide better imaging). NovaFISH probes are labeled with Atto488, Atto565, and Atto647N combinations.