Q
How to minimize tissue wrinkling during cryosectioning?
A
It is recommended that the cold mounting method be selected and solutions be added and discarded slowly throughout the process.
Q
Does omitting the primary and secondary antibodies, along with the Fc blocking reagent, during the permeabilization step affect the determination of the optimal permeabilization time?
A
No, it won't affect the results.
Q
If a fresh frozen sample has already been tested in the Stereo-seq Transcriptomics workflow with other staining methods (e.g., H&E or ssDNA), is it necessary to perform a permeabilization optimization experiment using the mIF protocol?
A
The permeabilization optimization experiment is necessary for the mIF workflow. In the mIF permeabilization protocol, the mIF staining and imaging process should take place between the methanol fixation and tissue permeabilization steps, although the primary and secondary antibodies, along with the Fc blocking reagent, can be replaced with nuclease-free water.
Q
Is the Fc blocking reagent required for the blocking solution?
A
It is strongly recommended that users add Fc blocking solutions that are not homologous to the primary antibody.
Q
Can I move the Stereo-seq Chip Slide after switching the channels for an mIF image?
A
Do not move the Stereo-seq Chip Slide when capturing multiple IF images. The slide must be kept still until the IF images from all channels have been captured. Moreover, applying a drop of water on the imaging stage prior to positioning the Stereo-seq Chip Slide is critical to the imaging process. This ensures the slide remains stationary during imaging, thereby avoiding any mobile bias.
Q
What is the best way to hadle areas of excessively high signal that impact judgment in the IF image?
A
There are two options.
Option 1: Avoid selecting fluorophores with overlapping wavelengths that may lead to spectral crossover;
Option 2: Replace a microscope to eliminate the risk of channel bleed through.
Q
Why is a brief centrifugation recommended before adding primary, secondary antibodies and DAPI solutions?
A
Brief centrifugation prevents the pipetting of antibodies or dye aggregates that have formed during storage, which can lead to bright spots on the fluorescence image.
Q
Can different antibodies be added in the mIF staining process? Which sources of antibodies have been tested so far?
A
The primary antibody should be mixed with different primary antibodies of different origins (primary antibody and secondary antibody should be of the same origin), and so far only primary antibodies of mouse/rabbit/rat origin have been tested.
Q
What points should be considered before and after drawing hydrophobic circles on the microscopic glass slide with an immunohistochemistry pen?
A
Before drawing the hydrophobic circle, ensure the slide around the tissue is dry. You can use dust-free paper to gently wipe away any moisture from the slide.
Hold down the slide with one hand and draw the circle to prevent it from moving.
When adding liquid, make sure to add it inside the hydrophobic circle, just enough to cover the entire tissue.
Avoid adding too much liquid, as it may overflow the hydrophobic circle, making it difficult for the circle to contain the liquid within.
Be careful when suctioning and disposing of the liquid; do not suction outside the hydrophobic circle.
When disposing of the liquid, ensure you do not drop any liquid outside the hydrophobic circle and keep it dry.
Q
When performing antibody titrations on microscopic glass slides, how many tissue sections can be mounted on one slide?
A
It is recommended to mount only one tissue section per microscopic glass slide to prevent the tissue from drying out during subsequent operations.
