We have tested that transferring tissue from -80℃ freezer to cryostat up to 4 times will not affect the mean gene counts in mouse brain tissue.

In addition to differences in tissue types, hole formation in the tissue may often result from the following reasons: (1) ice crystal formation due to slow or ineffective freezing; (2) air bubble introduced in freezing the tissue with OCT; (3) wrong type of brush was used that can damage fragile tissues.

If your cutting blade is not sharp enough, it may be helpful to change out your cutting blade. You can also 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.

First, check whether the equilibration time before sectioning is long enough. Too low of a specimen temperature might contribute to tissue cracking. Similarly, too low of a specimen head temperature in the cryostat can also lead to tissue cracking. In addition, too many ice crystals produced during the embedding, deficiencies in cutting blade or anti-roll plate can also cause tissue cracking.

The chip is not cleaned enough. Wash the chip with nuclease-free water for additional times.

Wrinkling of cryosections produced during mounting, tissue folding, and large sectioning thickness can all contribute to section detachment. Tissues that contain cavity structures will also increase the risk of detachment. Pre-coat the chip with PLL (Poly-L-lysine) is recommended to prevent section detachment. The following procedures could be tried during the chip preparation steps. 

Additional preparation reagent: Poly-L-lysine solution, 0.01% (P4707-50ML, SIGMA)
a. Transfer the chips stored in TE buffer to a new 24-well plate using forceps and record the chip number.
b. Gently add 400 μL of nuclease-free water along the wall of the well and pipette the liquid from the side without touching the surface of the chip.
c. Place the chip in a petri dish with a parafilm attached to the bottom, add poly-L-lysine solution dropwise (total of 50 μL) and incubate for 10 min at room temperature.
d. Aspirate the poly-L-lysine solution, transfer the chip to a 24-well plate, and add nuclease-free water to the 24-well plate (400 μL/well).
e. Carefully transfer the chip from the well plate to a dust-free paper with forceps to remove the water from the back side.
f. Hold the chip with forceps in one hand, fix it on the dust-free paper and slowly blow off the excess water with a power dust remover (MATIN, M-6318) from the side of the chip at a horizontal 30-45° angle.
g. Placed the chip on the slide dryer at 37℃ for 1 min. When the chip surface is without any liquid residue and without ripple-like texture, the chip is ready for tissue mounting.

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.

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-mix before use. We strongly recommend users to purchase and use the HCl from Sigma Aldrich (2104-50mL).

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).

There is high risk of RNA degradation so please arrange the experiment time accordingly and strictly follow the standard experiment procedure.