RNA Detection

1. Before initiating the staining, switch on the LCM system
   (microscope, laser, computer, start LMD software) and turn
   on the UV light and fan in the LCM chamber if available. Thaw
   slides for 1 min at room temperature by placing them horizon-
   tally on a clean surface (e.g., 5 mL pipette tips on plastic tray).


2. Place into acetone for 5 min (seeNote 8).


3. Incubate the slides three times in PBS (1 min each time) and
   subsequently dry the slides by briefly tilting these onto lint-free
   tissue. Note that after each step in the antibody staining proce-
   dure the solutions are removed and slides are dried briefly by
   simply tilting these onto lint-free tissue.


4. Place slides horizontally, cover with ~250μL, of primary anti-
   body staining solution for 4 min (seeNote 9), then remove
   staining solution.


5. Wash the slides three times in PBS (2 min each time) and
   subsequently dry briefly.


6. Place slides horizontally and cover the tissues with ~250μL
   secondary antibody staining solution for 4 min (seeNote 10),
   then remove staining solution.


7. Wash the slides three times in PBS (2 min each time) and
   subsequently dry the slides briefly.


8. Place slides horizontally, cover with ~250μL of ABC solution
   for 4 min, then remove staining solution.


9. Wash the slides three times in PBS (2 min each time) and
   subsequently dry the slides briefly.


10. Place slides horizontally, cover with ~250μL of DAB solution
    for 1–2 min, then remove staining solution.


11. Wash the slides two times in PBS (2 min each time) and
    subsequently dry briefly.


12. Wash in ddH 2 O for 30 s, then dehydrate the tissues by moving
    slides through rising ethanol concentrations (50, 75, 95, and
    99.7% ethanol, 30 s each) followed by briefly drying the slides.


13. Place slides into a dry, new slide box, air-dry the slides
    completely and initiate the LCM procedure.

3.5 Isolation of Cells
by Laser Capture
Microdissection

Perform LCM (as exemplified by using a Leica LMD7000, cells

captured by gravity,seeNote 11):

1. Turn off the UV light in LCM chamber.


2. Place the open slide box (with slides in a horizontal position)
   into the LCM chamber with the fan still running for 3–5 min to
   dry slides properly. Subsequently turn off the fan.

Spatial Transcriptomic Profiling Using LCM-seq 103