Q
What is the cause for serious distortion of the tissue during embedding?
A
The distortion of tissue could be caused by tissue compression during tissue harvest. In this case, tissue harvest method should be adjusted accordingly. The volume expansion of the OCT during freezing could also lead to tissue distortion. The embedding conditions should be adjusted according to the specific situation, such as, considering changing Cryomold or stainless-steel based mold size or material.
Q
How to avoid ice crystal formation within the tissue?
A
Excess liquid on the tissue should be removed before freezing. Rapid freezing is recommended to avoid ice crystals in the tissue since slow freezing produces large ice crystals.
Q
Will the amount of OCT affect the tissue during embedding?
A
Tissue may be squeezed when submerged in excess OCT, especially for small sized tissue. Therefore, it is recommended to choose an appropriately sized Cryomold or stainless-steel based mold
.
Q
Does OCT affect RNA extraction and RIN evaluation?
A
Yes, excessive OCT will affect the result of RNA extraction.
Q
How to avoid mRNA degradation during fresh tissue preparation?
A
It is strongly recommended to perform tissue embedding within 30 minutes after tissue harvest to avoid tissue RNA degradation to the greatest extent.
Q
What is the acceptable number of times for transferring tissue section from a-80℃ freezer to cryostat?
A
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.
Q
What is the cause for hole formation in cryosection?
A
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.
Q
How to resolve tissue curling during sectioning?
A
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.
Q
What is the cause for tissue cracking?
A
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.
Q
Why do white stains appear after the chip drying step?
A
The chip is not cleaned enough. Wash the chip with nuclease-free water for additional times.
