Question: Are fresh-frozen tissues containing bone compatible with Visium Spatial Gene Expression?
Answer: It was determined that fresh-frozen tissues containing bone require further sample preparation optimization for the Visium Spatial Gene Expression assay.
For less calcified tissues from earlier developmental time points such as developing bone from postnatal mice, our standard Tissue Preparation Guide for Visium for Fresh Frozen Tissues was shown to be successful in this customer publication. However, it is challenging to acquire high-quality data from tissue containing calcified bone.
Internally, we have tried two different methods for preparing bone tissue (adult mouse femur):
The first method we tested was to follow the standard Visium tissue freezing protocol. The fresh mouse femur was snap-frozen immediately after dissection and then embedded in OCT. The samples were subsequently cryosectioned using a tungsten carbide blade and collected on Visium slides. The standard conditions maintained the original anatomy but resulted in difficult tissue placement because calcified tissue is difficult to section.
- Undecalcified bone tissue is hard/brittle and tends to break apart during the sectioning process. The hard bone can also damage the metal blade in the cryostat, which increases the chances of tearing and/or compression of the surrounding non-bone tissue during sectioning. Therefore, if your tissue section contains bone, it will be difficult to get high-quality sections for the non-bone portions of the tissue due to damage to the blade.
- Following our standard Tissue Preparation Guide (freezing the fresh tissue and embedding in OCT), you may be able to get good gene expression data but getting good quality tissue sections will likely be challenging.
- The second method tested was to perform standard EDTA decalcification, which makes the tissue easier to section. The mouse femur was perfused with 4% PFA, decalcified using EDTA dissolved in 1x PBS for 1 week, and then sucrose-cryoprotected and OCT-embedded. The OCT-embedded tissue was cryosectioned and placed on Visium slides. While the anatomy was largely intact, the cross-linking from the aldehyde fixation impacted transcript quality and accessibility, leading to poor Gene Expression data.
Prior to processing bone tissues with the Visium Gene Expression assay, we would strongly recommend performing the following steps:
- Perform an RNA Quality Assessment on your tissue block to determine if the tissue has preserved RNA integrity (having tissue with a RIN score ≧ 7 is necessary to achieve high-quality gene expression data). Details on how to perform the RNA Quality Assessment can be found in our Tissue Preparation Guide.
- Practice sectioning bone tissues on a plain glass slide to determine optimal sectioning parameters before placing tissues onto a Visium Gene Expression slide. When sectioning, it may help to include additional tissue surrounding the bone region to improve structural integrity and tissue adhesion.
- Perform H&E staining of the sectioned samples to determine if the block has maintained tissue morphology. Being able to identify discrete morphological features is critical to being able to map spots back to regions of the tissue.
- Perform the Tissue Optimization (TO) experiment to determine the ideal permeabilization time for the Gene Expression workflow. Generating quality cDNA libraries is only possible if the RNA from the tissue is high-quality and there are no RT-inhibitors present. The TO experiment will test if the tissue preparation methods are compatible with the RT reaction.
Product: Visium for fresh-frozen