HuluFISH Kit Protocol – Whole Mount
Document: HuluFISH Kit Protocol – Whole Mount
Release date: 2019/01/01
Associated product: HuluFISH Kit
HuluFISH is industry’s first multiplexing single molecule FISH (smFISH) probe developed by PixelBiotech GmbH. HuluFISH 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 HuluFISH. The following protocols are included: HuluFISH staining.
Step 1: Probe preparation
Resuspend HuluFISH probe in 40 ul DNase/RNase Free water (i.e. DEPC treated water or commercial water aliquots for molecular biology use, for HuluFISH midi and maxi, use 200 ul and 500 ul water to resuspend).
TIPS: Facilitate the dissolution of the HuluFISH probe in water by tapping the tube several times. Alternatively, leave the tube on the bench at room temperature for 20 min. The HuluFISH probe should be stored at -20 degrees or lower. It is ok for repeated use by freezing-and-thawing the probe at room temperature. 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, tumorsphere, etc.) 4% formaldehyde in 1x PBS for overnight at 4 degrees in a 1.5 ml epi tube. Remove formaldehyde by centrifugation at 500-1000rpm 1min (alternatively allow the settle of the sample by gravity) and then wash once with 135mM Glycine in 1x PBS to quench residual formaldehyde for 10 min.
TIPS: Fixation could also be done with 4% paraformaldehyde in 1x PBS. Fixation time can be shortened into 10 min at the room temperature.
Step 3: Washing the residual formaldehyde
Wash once with 1x PBS to remove residual for 10 min. Remove the supernatant by spin off briefly (500-1000 rpm, 1min), then store the sample in 70% Ethanol at 4 degrees overnight. Then the embryo could store in 70% Ethanol at -20 degrees for several months.
Step 4: Washing the samples with HuluWash
Before hybridization, wash the coverslip with 2xSSC, 2M Urea (HuluWash) for 2 times, each time 10 min at room temperature.
TIPS: Washing steps could be done on a shaker to have better removal of residual ethanol.
Step 5: Staining with HuluFISH probe
Dilute 0.5 ul of HuluFISH probe in step 1 into 50 ul 1x HuluHyb solution (2xSSC, 2M Urea, 10% dextran sulfate, 5x Denhardt’s solution). Take the HuluFISH working solution into the sample tube. Hybridize at 37 degrees overnight. Optionally with shaking at 500 rpm.
TIPS: Cover with an aluminum foil to protect from the light. HuluFISH probe is stable over 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 HuluWash for 4 times, each time 10 min at 37 degrees with shaking.
TIPS: Washing can also be 2 times, 30 min each at 37 degrees. Or some PixelBiotech users have tried 4 degrees overnight washing. And the signal is still well maintained. In the last 5 minutes of the last washing step, add DAPI solution to have the final 1 ug/ml to stain nuclei.
Step 7: Mounting
Remove the residual buffer on the coverslip by dipping onto a clean tissue paper. Pipette 10 ul Prolong Gold/Glass mounting solution on a clean glass slide, then immediately cover the mounting solution with the 13mm coverslip having stained cells. Cell side should be again facing down. Allow the sample to cure for 24-48 hours at room temperature according to the instruction from Prolong Gold/Glass.
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). HuluFISH 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 is standing for mouse Gapdh HuluFISH probe with one Atto488, one Atto565, and one Atto647N. GAPDH-2G1R is standing for Human GAPDH gene with 2 Atto488 and one Atto647N.
Appendix 1: Recommended Reagents from other vendors