Q
How to resolve tissue curling during cyrosectioning?
A
You can adjust the anti-roll plate to flatten out the section. In addition, the relatively high cryostat chamber temperature compared to specimen head temperature also leads to tissue curling. Check the cryostat temperature and set appropriate chamber and specimen head temperature to avoid tissue curling.
Q
What is the cause for tissue cracking in cryosectioning?
A
First, check whether the equilibration time before sectioning is long enough. Unsuitably low temperature of the sample block might contribute to tissue cracking. Similarly, unsuitably low temperature of specimen head temperature in the cryostat could lead to tissue cracking. In addition, too many ice crystals produced during the embedding, broken cutting blade or faulty anti-roll plate can also cause tissue cracking.
Q
How to deal with scratches on the chip during experiments?
A
RNA molecules could not be captured anymore in scratched areas. Tissue mounting should avoid scratched areas on the chip. If the area is too large, we suggest switching to a new chip.
Q
What should be paid attention to in permeabilization mixture preparation?
A
Since permeabilization process is sensitive to the pH, please use freshly prepared 0.01N HCl (pH = 2). Do not vortex the Permeabilization Enzyme Mix. Pipette the mix before use. We strongly recommend users to purchase and use the HCl from Sigma Aldrich (2104-50mL).
Q
How should I arrange my experiments if the cryostat and slide dryer are not in the same room?
A
We strongly recommend performing drying immediately after sectioning. Please move your slide dryer to the same room and place it right next to the cryostat. Stereo-seq adapter and thermocycler/thermomixer could be used in replacement of a slide dryer. We strongly recommend users to process tissue sections and perform fixation in the same lab area and only transfer the chips to another location at the end of methanol fixation procedure (while the chips are submerged in methanol).
Q
What should I do if I cannot proceed with imaging immediately after fixation?
A
There is high risk of RNA degradation so please arrange the experiment time accordingly and strictly follow the standard experiment procedure.
Q
Is Stereo-seq Transcriptomics Set for fresh frozen samples compatible with other staining methods besides ssDNA staining?
A
Stereo-seq FF V1.3 is compatible with both ssDNA and H&E staining.
Q
What is the reason for the gradual dimming of fluorescence when adding focus points during imaging?
A
Long-term exposure of excitation light will cause partial fluorescence quenching.
Q
Does incomplete tissue removal affect the results?
A
A small amount of tissue on the chip has little to no effect on the Stereo-seq Transcriptomics Set. However, it can affect Stereo-seq Permeabilization Set. Incomplete tissue removal contributes to strong background during imaging, which affects the judgment of the optimal permeabilization time.
Q
Can the same tissue block be sectioned several times throughout multiple days?
A
Fresh frozen sample blocks can be sectioned for multiple times with a proper long-term storage at -80℃. Please note: It is still recommended to conduct sample QC for morphology and RIN quality assessment before each experiment.
