Biotechne ACD RNAscope Multiplex Fluorescent v2 Specification sheet

For Research Use Only. Not for Diagnostic Use MK-50 010 /Rev A/Draft Date 06192017
1
1. One day before fixation, seed cells in growth
medium on chamber slides at a density that will
allow cells to be 80–90% confluent at the time of
fixation.
1. Remove growth media, and dissemble chambers.
2. Submerge the slides in a Tissue-Tek®container with
200 mL of 1X PBS.
IMPORTANT! Do not let cells dry out at any time.
Always use enough solution to submerge all the
cells.
3. Remove 1X PBS, and add 10% Neutral Buffered
Formalin (NBF). Incubate at ROOM TEMPERATURE (RT)
for 30 MIN.
4. Remove NBF, and gently rinse slides with 1X PBS.
Repeat twice.
1. Remove 1X PBS wash, and replace with 50 mL 50%
EtOH. Incubate at RT for 5 MIN.
2. Remove 50% EtOH, and replace with 50 mL 70%
EtOH. Incubate at RT for 5 MIN.
3. Remove 70% EtOH, and replace with 50 mL 100%
EtOH. Incubate at RT for 5 MIN.
4. Remove 100% EtOH, and replace with fresh 100%
EtOH. Incubate at RT for 10 MIN.
NOTE: The slides can be stored in 100% EtOH
at -
-
20°C for up to 6 MONTHS.
1. Submerge slides in 70% EtOH. Incubate at RT for
2 MIN.
IMPORTANT! Do not let cells dry out at any time.
Always use enough solution to submerge all the
cells.
2. Remove 70% EtOH, and replace with 50% EtOH.
Incubate at RT for 2 MIN.
3. Remove 50% EtOH, and replace with 1X PBS.
Incubate at RT for 10 MIN.

1. Lay slides on the bench, and add ~5–8 drops of
RNAscope®Hydrogen Peroxide to cover the entire
section.
2. Incubate the slides at RT for 10 MIN.
3. Remove the RNAscope®Hydrogen Peroxide solution
from one slide at a time by tapping and/or flicking
the slide on absorbent paper. Immediately insert the
slide into a Tissue-Tek®Slide Rack submerged in a
Tissue-Tek®Staining Dish filled with distilled water.
4. Wash slides 3–5 times by moving the Tissue-Tek®
Slide Rack up and down in the distilled water.
5. Repeat Step 4 with fresh distilled water.
1. Draw 2–4 times around each well/circle on the
chambered slides using the Immedge™hydrophobic
barrier pen. Let the barrier dry completely ~1 MIN.
NOTE: Do not let the cells dry out during this step.
Place slides back into 1X PBS if the cells look too
dry.
2. Rinse slides briefly with 1X PBS in a Coplin jar or
staining dish.
1. One at a time, remove each slide from the 1X PBS
and tap/flick to remove excess liquid. Place the
slides on the HybEZ™ Slide Rack, and place rack in
the Humidity Control Tray.
2. Add 2–4 drops diluted Protease III to completely
cover each well/circle.
NOTE: For most cell lines, dilute protease 1:15 with
1X PBS. Protease dilution factor must be empirically
determined for each new cell type.
3. Close the Humidity Control Tray and incubate for 10
MIN at RT.
4. One at a time, take each slide from the HybEZ™
Slide Rack and tap/flick to remove excess liquid.
Submerge slides in 1X PBS.
5. Wash the slides by agitating them in the 1X PBS.
Repeat with fresh 1X PBS.
IMPORTANT! Proceed to the RNAscope®protocol using
the R
RNAscope®Multiplex Fluorescent Kit User Manual
Part 2
(Catalog No. 323100) available at
https://acdbio.com/technical-support/user-manuals.
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