Question: What should I do if my FFPE tissue has detached from the Visium Gene Expression Slide?
Answer: FFPE tissue sections can be prone to detachment during on-slide workflows. When performing the Visium for FFPE assay, occasionally, tissue detachment may occur. This detachment can come in different severities: minor, major, or complete detachment (shown below).
If tissue detachment occurs before Permeabilization during the Probe Release step of the Visium for FFPE assay, there may be a negative impact on assay performance.
- We recommend not proceeding with the workflow when tissue has detached. However, if it is a precious sample and detachment is minor, it may be possible to continue with the protocol and generate spatial gene expression data. Keep in mind that the data in the detached region of the tissue section may be unusable in this case.
- As part of our replacement policy, detachments on Gene Expression Slides that have been properly documented are replaced with replacement reagents and consumables. Please provide the following information to firstname.lastname@example.org:
- An image of the Visium Slide (before and after detachment)
- The Visium Slide Serial Number (SN) and Slide Kit Lot Number
- Protocol step in which detachment occurred
- Number of capture areas and slides used
- Number of capture areas that experienced tissue detachment and the severity of the detachment
- Staining method used
- Tissue type and species
- Your shipping address (or a recent 10x purchase order) and a contact phone number
Best practices to minimize tissue detachment:
- Clean the workstation with RNase decontamination solution and 100% ethanol before use.
- Use a new, clean microtome blade for sectioning.
- Allow the tissue block sufficient time in the ice bath to ensure proper hydration. Sections from properly hydrated tissue should be shiny, transparent, and not opaque or dry.
- After hydration, collect the sections as soon as possible without allowing the block to warm up, or its surface to dry. Always discard the first section but collect sections very close to the surface, as sections deeper in the block are less hydrated and warmer. When collecting consecutive sections, rehydrate the block to obtain the best sections as soon as the sections start to come out opaque and dry.
- Confirm that the tissue block is without cracks, and the plastic cassette can firmly support the wax and the tissue. Unstable blocks will generate sections that are suboptimal.
- Ensure the microtome is set up properly so sections of uniform thickness can be generated.
- Section tissue at 5 µm.
- The water bath should be filled with Ultrapure/MilliQ water and be free of bubbles or particulates.
- Allow sections to flatten and stretch on the water bath. Visually inspect the sections and only collect the sections free of wrinkles, folds, tears, cracks, air bubbles, brittle edges, or other imperfections.
- Before collection onto Visium Slides, QC the tissue section with a plain glass slide to determine the optimal float time.
- If a section starts to disintegrate in the floatation bath after several seconds, try to collect it as soon as possible to ensure that it remains intact. Disintegration of a section is a sign of inadequate paraffin infiltration during tissue processing.
- Sections from the same tissue sample should be uniform in thickness, size, and shape. Thicker sections will have darker H&E staining.
- Properly dry sections by following the recommendations in our documentation.
- Gently add and remove reagents along the side of the wells of Slide Cassette during the workflow.
Products: Visium for FFPE