HuluFISH Kit Protocol – Tissue + HuluCLEAR


Document: HuluFISH Kit Protocol – Tissue + HuluCLEAR

Version: 0.3

Release date: 2019/01/01

Associated product: HuluFISH Kit, HuluCLEAR

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. 


When your tissue samples have an intrinsic high autofluorescence background, HuluCLEAR is recommended to alleviate the background problem by washing out small molecule (like NAD, etc.) derived fluorescence. HuluCLEAR also reverses the formaldehyde over crosslinking to make your RNA targets more accessible to HuluFISH probes.

This document is for using HuluFISH and HuluCLEAR with tissue. The following protocols are included: tissue section preparation, tissue clearing, and HuluFISH staining.


Step 1: Prepare tissue block
For the cryosectioned sample, snap-freezing your tissue sample as quickly as possible in O.C.T on dry ice (-78 degree, in 3 minutes), or directly in liquid nitrogen equilibrated 2-methyl butane (around -150 degree, 1 minute). Store the sample in -80 degree before cutting in the cryostat. For FFPE samples, standard fixation with formalin and embedding with paraffin will be acceptable for HuluFISH. Store the tissue block in paraffin at -20 or -80 degree.

TIPS: HuluFISH can work with cryo-sectioned frozen tissues or FFPE (formalin-fixed paraffin-embedded) sections. For best RNA quality, tissue blocks should always be stored at low temperature to prevent chemical degradation and RNase mediated degradation of RNA. 

Step 2: Sectioning tissue block and mounting
Cut your tissue block with 3-5 um thickness for best HuluFISH staining. Mount them onto our special treated 13 mm coverslips (HuluCoverslip). For FFPE sections, the sample is required to be further baked on 37-degree hot plate for an additional 2 hrs for strong binding to the coverslips. For cryosections, cutting should be done at -20 degree in a cryostat (i.e. cryostat from Leica). 

TIPS: HuluFISH can work with thicker tissue, like 10-20 um, or even whole mount embryo of platynereis (100 um thick). However thinner tissue sections usually give less background and fewer diffraction artifacts during image acquisition on a microscope.

TIPS: For cryosectioned tissues, the coverslips should be kept at room temperature for efficient binding with the thin tissue cryosections. When tissues are mounted onto coverslips, these coverslips either should be directly transferred into a 24-well cell culture plate on dry ice or left in the cryostat (around -20 degree) until all tissue sections finished and then transferred into the 24-well plate on dry ice. All sections should be stored at -80 degree before use. Both sides of the coverslip are specially treated to adhere to the tissue easily and allow following clearing steps.


TIPS: For transportation of these unfixed cryosections to another place, the whole 24-well plate should be sealed properly (with tape) and kept in a box with sufficient dry ice for the whole transportation process. For FFPE sections, they can be transported in 24-well plate at room temperature.


Step 3a: Fixation (optional for cryosections)
Unfixed cryosections should be fixed in 4% formaldehyde in 1xPBS for 10 min. Then quenching the fixation reaction in 135mM Glycine in 1xPBS for another 10 min. Then another 1xPBS washing for 10 min is required to remove residual formaldehyde and glycine. Finally, the sample should be permeabilized in -70% ethanol overnight at 4 degree.

TIPS: All buffers prepared here and after should be DEPC treated.


TIPS: After this fixation step, the RNA in cryosection will be more stable and becoming a bit more resistant to RNase, but still vulnerable to heat-induced chemical degradation of RNA. Therefore the fixed cryosection should still be stored in 70% ethanol at -20 degree. The RNA will be stable from several months to a year. 
Cryosection sample may be appropriate for transportation at this step. Transportation should be -20 degree with enough 70% ethanol or even higher concentration ethanol. Again the 24-well plate should be well sealed with tape.

Step 3b: De-paraffinization (optional for FFPE sections)
Wash the paraffin sections on coverslips with Xylene for 2 x 1 hrs. Then wash out the residual Xylene with 100%, 95%, 70% ethanol gradient, each step 10 min.

TIPS: Longer deparaffinization (2hrs in total) in xylene removes paraffin maximally hence reduce the background staining. Even longer xylene treatment will destroy the sample on the coverslips.




Step 1: Acrylamide modification
Rehydrate tissue sections in 1xPBS for 10 min. Then modify the sample with acrylamide and formaldehyde in 30% acrylamide, 4% formaldehyde in 1x PBS for 2 hour at room temperature. Then wash the sample with 1x PBS for 2 x 10 min. 

TIPS: The modification solution should be prepared freshly. For example, mixing 7.5 vol of 40% acrylamide, 1 vol of 37% formaldehyde, 1 vol of 10xPBS, 0.5 vol of DEPC treated water.


Step 2: Gel embedding
Incubate the sample with 10 ul HuluCLEAR ready gel solution for 30 min at room temperature. Then take another 10 ul ready gel solution on the hydrophobic glass slide, cover the solution with coverslips with the sample facing down to the solution. Polymerize under blue LED (minimal 40W) for 120 s.

TIPS: HuluCLEAR ready gel solution is a proprietary solution from PixelBiotech GmbH. The hydrophobic glass slide is also provided by PixelBiotech GmbH. Blue LED light source should be at least 20W. 


Step 3: SDS clearing
Wash the gel-embedded sections in 2xSSC for 10 min. Then clear in 8% SDS in 2xSSC at 37 degrees in a 5 ml epi tube. Shake the tube on heat block with 500 rpm for 2 hrs.

TIPS: Longer clearing with SDS does not improve background reducing but reduce the signal by facilitating the RNA elution from the gel.




Step 1: Probe preparation
Resuspend HuluFISH probe in 33 ul DNase/RNase Free water (i.e. DEPC treated water or commercial water aliquots for molecular biology use).

TIPS: Facilitate the dissolution of 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 degree or lower. It is ok for repeated use by freezing-and-thawing the probe at room temperature. All water used in following steps to prepare the buffer should also be DEPC treated to minimize the RNase contamination.


Step 2: Washing the sections with HuluWash
Before hybridization, wash the coverslip with HuluWash (2xSSC, 2M Urea)  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 3: Staining with HuluFISH probe
Dilute 0.5 ul of HuluFISH probe in step 1 into 50 ul 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 2. Remember the side with tissues should face down to the solution. Hybridize in a humidified chamber at 30 degree for overnight.

TIPS: Staining time is minimally 4 hours with HuluFISH. Usually, overnight staining guarantees sufficient staining but also a convenient step for a break in your experiment. 


Step 4: Washing the unbound probe
Wash the coverslip in the culture well with 2xSSC, 10% formamide, 0.1 % Tween-20 (WashT) for 4 times, each time 10 min at room temperature. Add 1/200 vol of the 200x DAPI solution in the last 5 min of washing.

TIPS: Washing can also be 2 times, 30 min each at room temperature. Or some PixelBiotech users have tried 4 degree overnight washing. And the signal is still well maintained.

Step 5: 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. Tissue 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: The sample could also be mounted in 2xSSC for immediate imaging.

Imaging the target by epifluorescence or confocal microscope with an appropriate laser. 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

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

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

  • 40% Acrylamide, Bio-Rad, Catalog Number 1610140 (link)