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HuluFISH Kit Protocol – Immunofluorescence Staining

INTRODUCTION

Document: HuluFISH Kit Protocol – Immunofluorescence Staining

Version: 0.3

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. 

 

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

IMMUNOFLUORESCENCE STAINING

Step 1: Cell fixation
Cultivate cells on 13 mm #1 coverslip. Fix the cells in 3% PFA/PBS, room temperature for 10 min. Then wash with 1ml PBS at room temperature for 1 min. Then wash with 1ml 0.5% Triton-100/PBS at room temperature for 10 min. Then 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 4 times PBS 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: HuluFISH staining will immediately follow the IF staining in order to preserve signal maximally.

 

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

 

TIPS: If the coating is required for your cell line, coat the coverslip with a molecule required for your cell to attach and grow. Before fixing the cell with 4% formaldehyde, cells could grow at the various confluence (10-100%), but at least one overnight to allow enough stickiness of cells to the coverslip. Fixation could also be done with 4% paraformaldehyde in 1x PBS. Normally we use 12 or 24 well cell culture plates for cultivation, fixation, and the washing steps.

 

Step 3 Washing the residual formaldehyde
Wash once with 1x PBS to remove residual for 10 min. Then store the sample in 70% Ethanol at 4 degrees overnight. Then the cells could store in 70% Ethanol at -20 degrees 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 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 on a clean piece of parafilm, then cover the solution with washed coverslip from step 4. Remember the side with cells should face down to the solution. Hybridize in a humidified chamber at 30 degrees overnight.

TIPS: The cell side of coverslip can be easily distinguished if you look at the coverslip under the light. The cell side is less reflective than the non-cell side. Or experimenter could also check the coverslip on the light microscope and try to scrape some cells from the upside of the coverslip. If there is no cell that can be scraped off, the upside is the non-cell side.

 

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

 

TIPS: Washing can also be 2 times, 30 min each at room temperature. 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 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)