Question: Is Immunofluorescence (IF) staining compatible with Fixed Frozen mouse tissue samples?
Answer: Based upon limited testing performed with three tissue types, Fixed Frozen (FxF) mouse tissue samples are compatible with IF staining. In this article, we will describe the workflow steps and modifications required to successfully execute IF staining with mouse FxF samples.
Prior to carrying out the Fixed Frozen IF Staining workflow to generate sequencing ready libraries, ensure antibody(s) are compatible with Fixed Frozen tissue sections and concentration for staining has been optimized following guidance provided in the Antibody Optimization section of the Visium CytAssist Spatial Gene Expression for FFPE – Deparaffinization, Decrosslinking, Immunofluorescence Staining & Imaging Demonstrated Protocol (CG000519). When performing Antibody Optimization, ensure Steps A - D as outlined below are followed for compatibility with Fixed Frozen tissue sections.
Once Antibody Optimization has been completed, use dilutions of compatible antibody(s) determined to execute the Fixed Frozen IF staining workflow through library construction as outlined in Steps A - E below.
Step A: Prepare Tissue Slide(s) following the Visium CytAssist Spatial Gene Expression for Fixed Frozen – Tissue Preparation Guide (CG000663) without modification.
Step B: Rehydrate Tissue (Step 1.2), following the Visium CytAssist Spatial Gene Expression for Fixed Frozen – Rehydration, H&E Staining, Imaging & Decrosslinking Demonstrated Protocol (CG000662) with the following modifications:
- DO NOT proceed to H&E staining once you reach the end of the rehydration step (Step 1.2h).
- Ensure to move immediately to the Decrosslinking step to avoid over drying of tissue sections.
Step C: Decrosslink (Step 1.3) following the Visium CytAssist Spatial Gene Expression for FFPE – Deparaffinization, Decrosslinking, Immunofluorescence Staining & Imaging Demonstrated Protocol (CG000519) with the following modification:
- Perform Decrosslinking at 70C for 30 min, NOT 95C for 1 h as indicated.
Step D: Immunofluorescence Stain (Step 1.4 A or B); depending on if performing direct or indirect staining, respectively, Coverslip (Step 1.5), Image (Step 2.2), & Decoverslip (Step 2.3) following the Visium CytAssist Spatial Gene Expression for FFPE – Deparaffinization, Decrosslinking, Immunofluorescence Staining & Imaging Demonstrated Protocol (CG000519) without modification.
Step E: Construct Library (Steps 1-5) following the Visium CytAssist Spatial Gene Expression Reagent Kits User Guide (CG000495) without modification.
Sections from three mouse Fixed Frozen tissue types: brain, thymus, & kidney were H&E or IF stained using fluorophore conjugated primary antibodies (direct). Visium CytAssist Spatial Gene Expression probe-based libraries were constructed, sequenced, and the data was compared (Figure 1).
For Mouse Brain, we observed morphological differences between sections stained with H&E compared to IF. This can explain the slightly higher sensitivity: Genes per Spot ~10% and UMI Counts per Spot ~20% in the IF stained samples. However, Mouse Thymus & Kidney sections showed similar morphology between H&E and IF sections and no difference was observed when comparing Gene and UMI Counts per Spot. Overall, the three Fixed Frozen mouse tissue types tested yielded comparable assay sensitivity irrespective of the staining method tested.
Figure 1. Each bar in Figure 1 represents data from two sections. All changes in UMI counts where downsampled to the same sequencing depth of 10,000 read pairs per tissue-covered spot.
Product: Visium CytAssist for FxF