NovaFISH Kit Protocol – Whole Mount

INTRODUCTION

Document: NovaFISH Kit Protocol – Whole Mount

Version: 1.4

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 cell, fixed tissue sections, or fixed whole mount embryo. 

This protocol is for the whole mount staining of RNA in embryo/tumorsphere by NovaFISH. The following protocols are included: NovaFISH staining.

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 dissolution 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 freezing-thawing cycles of the probe do not affect the product. All water used in the following steps to prepare the buffer 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 Sample Preparation
Fix with sample (whole embryo, tumor sphere, etc.) formaldehyde 4% in 1x PBS overnight at 4 ?C in a 1.5 mL epi tube. Remove formaldehyde by centrifugation at 500-1000 rpm 1 min (alternatively, allow the settle of the sample by gravity) and then wash once with Glycine 135 mM in 1x PBS to quench residual formaldehyde for 10 min.

TIPS: Fixation could also be done with paraformaldehyde 4% in 1x PBS. Fixation time can be shortened to 10 min at room temperature.

Step 3: Washing the residual formaldehyde
Wash once with 1x PBS to remove residual for 10 min. Remove the supernatant by spinning off briefly (500-1000 rpm, for 1 min), then store the sample in ethanol 70% at 4 ?C overnight. Then the embryo could store in ethanol 70% at -20 ?C for several months.

Step 4: Washing the samples with NovaWash
Before hybridization, wash the coverslip with NovaWash (2xSSC, Urea 2 M) twice, each for 10 min 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). Take the NovaFISH working solution into the sample tube. Hybridize at 37 ?C overnight. Optionally with shaking at 500 rpm.

TIPS: Cover with an aluminum foil to protect from the light. NovaFISH probes are stable under ambient light. However, long-time exposure to strong light is detrimental to the probe quality.

Step 6: Washing the unbound probe
Wash the coverslip in the culture well with NovaWash for 4 times, each for 10 min at 37 ?C with shaking.

TIPS: Washing can also be 2 times, 30 min each at 37 ?C. Some PixelBiosciences users have tried washing at 4 ?C overnight, 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 on a clean tissue paper. Pipette 10 μL of mounting medium on a clean glass slide, then immediately cover the mounting solution with the 13 mm coverslip having stained cells facing down. Store the sample at 4 ?C for a couple of hours before 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 Human GAPDH gene with 2 Atto488 and one Atto647N.
 

Appendix

Appendix 1: Recommended Reagents from other vendors

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

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