- Embedding medium (OCT).
- Embedding molds.
- Block of dry ice.
- Forceps, precooled on dry ice.
- Razor blade or scalpel to trim tissue block.
- Slide box.
- LCM membrane slides (e.g., 1.0 PEN membrane glass slides,
Zeiss). - Pencil to label slides, permanent marker to label embedding
mold. - Brushes to handle tissue sections and cleaning of blade.
2.3 Tissue Staining
and LCM
- Leica LMD7000 (any LMD microscope can be used, but
depending on the vendor it might change which microscope
slides and collector tubes are used), preferably enclosed in a
clean chamber equipped with a fan and a UV lamp. - Thermometer, hygrometer.
- RNaseZap wipes.
- One styrofoam box with dry ice and one with wet ice.
- Staining containers (e.g., conventional 50 mL centrifuge
tubes): 2ddH 2 O (for HistoGene staining only), 275%
ethanol, 195% ethanol, and 199.7% ethanol, 25 mL each. - Pipette boy, clean; 25 mL pipette tips, sterile.
- 99.7% ethanol.
- Nuclease-free ddH 2 O.
- HistoGene staining solution (Arcturus) or 1% cresyl violet
acetate solution in 75% ethanol adjusted to pH 8.0 with 3 M
Tris–HCl. - Plastic staining tray, 5 mL pipette tips, sterile, to place slides on.
- Lint-free tissue wipes.
- Slide forceps.
- Slide box.
- Pipettes and filter tips (1–10μL and 200μL).
- Timer.
- Lysis buffer: 0.2% Triton X-100, 2 U/μL RNase inhibitor, kept
on ice, prepared freshly for every session. - 0.2 mL RNase/DNase-free PCR tubes (seeNote 11).
- Microcentrifuge (e.g., VWR Ministar silverline).
- Parafilm.
Spatial Transcriptomic Profiling Using LCM-seq 97