Question: How do I determine the optimal permeabilization time from my Visium Tissue Optimization (TO) experiment?
Answer: Several key considerations should be taken into account before, during, and after the Tissue Optimization (TO) experiment to determine the optimal permeabilization timepoint for your tested tissue type and section thickness.
Before the Tissue Optimization experiment:
A high-quality tissue block should be used to generate serial sections of the same thickness. These sections should be from the same tissue region that will be processed during the Gene Expression assay. Once sections are placed onto the TO slide, fix, and stain the tissue following the appropriate Demonstrated Protocol (DP) (H&E or IF). After staining, image all capture areas in a single, stitched image, ensuring that sections are in focus. Please refer to our Imaging Guidelines Technical Note for more details.
During the Tissue Optimization experiment:
An appropriate permeabilization time course and having (positive and negative) controls are critical for determining the optimal permeabilization conditions. A longer time course (0-60 minutes) may be required for certain tissue types than standard (0-30 minutes); please consult our Visium Spatial Gene Expression Optimized Tissues webpage. This webpage lists tissue types, section thickness, and permeabilization time course tested in-house. After the permeabilization time course and Tissue Removal step, ensure that all tissue is removed and acquire a single, stitched image of all capture areas using fluorescent settings (TRITC). During imaging of cDNA footprint:
- Ensure images are in focus
- Out-of-focus images can interfere with image analysis.
- Zoom-in on the fluorescent image to check the focus.
- If out of focus, retake the image.
- Image Capture Areas using the same settings and proper exposure.
- Over- or under-exposure can interfere with the image analysis.
The negative (no permeabilization) control should show minimal fluorescence signal, while the positive (no tissue, bulk RNA) control should generate a signal where RNA was spotted on the slide. The negative control will help determine background fluorescence, while the positive control verifies that the reverse transcription reaction was successful.
To assess permeabilization times, zoom-in. The permeabilization time that generates the brightest and crispest fluorescent cDNA footprint showing the least amount of signal outside the tissue is the condition that should be used for the Visium Spatial Gene Expression experiment. This experiment's optimal time point is designated with a white box (18 minutes; right image). At 18 minutes, the cDNA footprint is bright and sharp (hippocampal regions; outlined in the left image), and the tissue edge is intact (arrow in left image).
Please note: This experiment excluded a positive control only for demonstration purposes.
Below is an example of a Visium TO experiment with human breast cancer tissue, including the brightfield H&E (left) and TRITC (right) images. Tissue sections should be high-quality and properly placed within the capture area; this is shown in the brightfield image. This experiment's optimal time point is designated with a green checkmark (12 minutes; right image).
If the fluorescent signal is comparable at two-time points (6 and 12 minutes in the above example), select the longer time point. Furthermore, the optimal permeabilization time will depend on tissue type, sample quality, section thickness, user, staining method, tissue region of interest, and other variables. Additional examples of Tissue Optimization experiments can be found in the Imaging Guidelines Technical Note.
After the Tissue Optimization experiment:
For the optimal permeabilization time determined during the TO experiment that will be applied to the Gene Expression assay, please ensure that the following conditions are consistent between experiments:
- Tissues used in the TO and Gene Expression experiments should have a similar, high RNA Integrity Number (RIN). To determine RIN scores, perform an RNA Quality Assessment on the tissue block(s).
- Tissue with a RIN score of ≧7 is necessary to achieve optimal assay performance.
- Use the same tissue block or one with the same sample preparation, a similar region of interest, same section thickness, and same staining method.
- Differences in any of the above variables may lead to suboptimal permeabilization during the Gene Expression experiment.
- Optimal permeabilization times may differ between healthy and diseased tissue.
- Separate TO experiments should be performed.