NovaFISH Kit Protocol – Immunofluorescence Staining

INTRODUCTION

Document: NovaFISH Kit Protocol – Immunofluorescence Staining

Version: 0.4

Release date: 2024/03/05

Associated product: NovaFISH Kit

NovaFISH is the industry’s first multiplexing single molecule FISH (smFISH) probe and was developed by PixelBiosciences GmbH. NovaFISH can be used to detect DNA/RNA expression in digital quantification and the sample can be in isolated DNA/RNA, fixed cells, fixed tissue sections, or fixed whole mount embryo. 

NovaFISH can also be combined with immunofluorescence staining (IF). This manual is for the combined detection of RNA with NovaFISH and immunofluorescence staining in fixed cells. The following protocols are included: immunofluorescence staining and NovaFISH staining.

IMMUNOFLUORESCENCE STAINING

Step 1: Cell fixation
Culture cells on 13 mm #1 coverslip. Fix the cells with PFA/PBS 3% at room temperature for 10 min. Then, wash with 1 mL of PBS at room temperature for 1 min. Continue with another washing step with Triton-100/PBS 0.5% at room temperature for 10 min. Last, wash once with PBS for 1 min at room temperature.
 

Step 2: Primary antibody incubation
Add 1st antibody and incubate at room temperature for 1 hr. Then wash with PBS 4 times for 5 min each at room temperature. 
 

Step 3: Secondary antibody staining
Add 2nd antibody and incubate in dark at room temperature for 1 hr. Then wash with PBS 3 times for 5 min each at room temperature.

TIPS: NovaFISH staining will immediately follow the IF staining to preserve maximal signal.

NovaFISH STAINING
 

Step 1: Probe Preparation
Reconstitute NovaFISH probe nano in 10 μL DNase/RNase Free water (i.e. DEPC treated water or commercial water aliquots for molecular biology use). For NovaFISH mini use 40 μL, midi 200 μL, and maxi 500 μL water to reconstitute).

TIPS: Facilitate the reconstitution of the NovaFISH probe in water by tapping the tube several times. Alternatively, leave the tube on the bench at room temperature for 20 min. The NovaFISH probe should be stored at -20 ?C or lower. Repeated cycles of freezing and thawing do not affect the product. All water used in the following steps to prepare the buffers should also be DEPC treated to minimize the RNase contamination (for a detailed protocol of how to make DEPC treated solution, check https://en.wikipedia.org/wiki/Diethyl_pyrocarbonate).

Step 2: Cell Preparation
Fix the antibody-stained cells (see previous section) with formaldehyde 4% in 1x PBS for 10 min at room temperature. Remove formaldehyde and then wash once with Glycine 135 mM in 1x PBS to quench residual formaldehyde for 10 min. 

TIPS: If coating is required for your cell line, coat the coverslip with the reagent required for your cells of interest to attach and grow. Before fixing the cell with formaldehyde 4%, cells can be allowed to grow at various degrees of confluence (10-100%), but at least overnight to allow enough cell adherence to the coverslip. Fixation could also be done with paraformaldehyde 4% in 1x PBS. Usually, 12- or 24-well plates are used for cell culture, fixation, and washing steps.

Step 3 Washing the residual formaldehyde
Wash once with 1x PBS to remove residual for 10 min. Then store the sample in ethanol 70% at 4 ?C overnight. Then the cells can stored in ethanol 70% at -20 ?C for several months.

TIPS: Seal the culture well plate containing the fixed cells on the coverslip with parafilm or tape to minimize the evaporation of ethanol. Or regularly check the ethanol level in the well to make sure there is no dry-out of the cells.

Step 4: Washing the cells with NovaWash
Before hybridization, wash the coverslip with NovaWash (2xSSC, Urea 2M) twice for 10 min each at room temperature.

TIPS: Washing steps could be done on a shaker to better remove residual ethanol.

Step 5: Staining with NovaFISH probe
Dilute 0.5 μL of NovaFISH probe in step 1 into 50 μL 1x NovaHyb solution (2xSSC, Urea 2M, dextran sulfate 10%, 5x Denhardt’s solution). Prepare a new and clean glass slide by making a circle of glue suitable for the purpose. Pipette into it 10 μL of probe working solution. Place the coverslip on top of the droplet of NovaFISH probe working solution with the cells facing down. Seal the preparation by adding more glue on top and around the edges of the coverslip. Hybridize in a humidified chamber at 30 ?C overnight.

TIPS: The side of the coverslip with cells can be easily distinguished by looking at the coverslip under the light. The cell-cultured side is less reflective than the cell-clear side. The user could also check the coverslip under the light of a microscope and try to make a very little scratch on top of the coverslip and check if some cells came off.

Step 6: Washing the unbound probe
Wash the coverslip in the culture well with NovaWash 4 times for 10 min each at room temperature.

TIPS: Washing can also be done twice for 30 min each at room temperature. Some customers of PixelBiosciences have tried 4 degrees overnight washing. And the signal is still well-maintained. Counter stains (such as DAPI) can be added to the washing step, preferably the first, and let the excess be washed away with the subsequent steps.

Step 7: Mounting
Remove the residual buffer on the coverslip by tapping gently onto a clean tissue paper. Pipette 10 μL of mounting medium on a clean glass slide, then immediately place the coverslip on top, with the hybridized cells facing down. Store the preparation at 4 ?C until use.

TIPS: Imaging the sample on the coverslip by epifluorescence or confocal microscope with an appropriate laser (newer model of confocal microscope usually has better imaging outcome). NovaFISH probe is labeled with the combination of Atto488, Atto565, and Atto647N. All fluorophores are barcoded as G (Atto488), Y (Atto565), and R (Atto647N). Check your probe barcoding scheme on the tube label. For example, Gapdh-1G1Y1R stands for mouse Gapdh NovaFISH probe with one Atto488, one Atto565, and one Atto647N. GAPDH-2G1R stands for the Human GAPDH gene with 2 Atto488 and one Atto647N.
 

Appendix

Appendix 1: Recommended Reagents from other vendors

Prolong Gold, ThermoFisher Scientific, Catalog Number P10144 (link)

Prolong Glass, ThermoFisher Scientific, Catalog Number P36982 (link)