RNAscope™ Multiplex Fluorescent v2 Assay for Cultured Adherent Cells in 96-well Plate Format

Introduction

This Technical Note provides guidelines for sample collection and pretreatment of cultured adherent cells that can be assayed using the RNAscope™ Multiplex Fluorescent Kit v2 (Cat. No. 323100). To pretreat the cells, use RNAscope Protease III (available in the RNAscope Protease III and Protease IV Reagents, Cat. No. 322340 or RNAscope Universal Pretreatment Kit Cat No 322380). Read the Safety Data Sheet (SDS) available on the website, and follow handling instructions. Wear appropriate protective eyewear, clothing, and gloves. For the latest services and support information, contact our Technical Services.

Workflow

Part 1: Cell Preparation

Cell Culture

1. One day before fixation, seed cells in growth medium on a 96-well plate at a density that will allow cells to be 80–90% confluent at the time of fixation.

NOTE:  Cells must be firmly adhered to the plate before starting the assay.

Cell Fixation

1. Remove growth medium, and wash with 200 µL/well of 1X PBS. Repeat wash step once.

IMPORTANT! Do not let cells dry out at any time during the assay procedure.

1. Remove 1X PBS, and add 200 µL/well of 10% Neutral Buffered Formalin (NBF). Incubate at ROOM TEMPERATURE (RT) for 30 MIN.
2. Decant NBF into a waste container, and gently rinse plate with 200 µL/well of 1X PBS. Repeat wash step twice.

Dehydrate and Store Cells

1. Remove 1X PBS wash, and add 200 µL/well of 50% EtOH. Incubate at RT for 1 MIN.
2. Remove 50% EtOH, and add 200 uL/well of 70% EtOH. Incubate at RT for 1 MIN.
3. Remove 70% EtOH, and add 200 µL/well of 100% EtOH. Incubate at RT for 1 MIN.
4. Remove 100% EtOH, and replace with 200 µL/well fresh 100% EtOH.

NOTE:  Cover 96-well plate with lid and seal with parafilm. Polystyrene bottomed plates can be stored at   –20ºC for up to 3 MONTHS. Glass bottomed plates can be stored at 4ºC for up to 48 HRS.

Rehydrate Cells

1. Remove 100% EtOH, and add 200 µL/well of 70% EtOH. Incubate at RT for 1 MIN.
2. Remove 70% EtOH, and add 200 µL/well of 50% EtOH. Incubate at RT for 1 MIN.
3. Remove 50% EtOH, and add 200 µL/well of 1X PBS. Incubate at RT for 1 MIN.
4. Remove 1X PBS, and add 200 µL/well of fresh 1X PBS. Incubate at RT for 10 MIN.

NOTE:  During the last incubation, prepare materials for Part 2 and pre-warm Target Probes to 40ºC.

Part 2: RNAscope Pretreatment

Prepare Materials

1. Make 3L 1X Wash Buffer by adding one bottle of 10X Wash Buffer (300 mL) and 2.7 L distilled water into a bottle or beaker.
2. Bring Hybridization Oven to 40°C.

NOTE:  Plate should be inserted inside the HybEZ^™ Oven without a Humidity Control Tray.

1. Bring RNAscope Multiplex Fluorescent Reagent Kit v2 to RT.
2. Prepare Mixed Target Probes by adding C1, C2, and C3 Probes in a tube at 50:1:1 volume ratio.

Apply RNAscope Hydrogen Peroxide

1. Keep the 96-well plate on the bench and add ~3 drops of RNAscope Hydrogen Peroxide to each well. Make sure to completely cover the cells.
2. Incubate the 96-well plate at RT for 10 MIN.
3. Remove the RNAscope Hydrogen Peroxide solution from the plate by inverting and gently tapping the plate on absorbent paper.
4. Wash each well with the fresh distilled water. Make sure each well contains liquid.
5. Repeat the wash step with fresh distilled water.

Protease Digestion

1. Remove excess liquid. Place the plate in the HybEZ^™ Humidity Control Tray.
2. Add 3 drops of a 1:15 dilution of Protease III to each well. Incubate at RT for 10 MIN.

NOTE:  For most cells lines, dilute Pretreat 3 to a 1:15 ratio with 1X PBS (for example, 20 µL Protease III with 280 µL 1X PBS). The exact protease dilution factor must be empirically determined for each new cell type.

1. Remove diluted Protease III solution, and add approximately 200 µL/well of 1X PBS. Incubate at RT for 2 MIN.
2. Repeat the wash step twice.

Hybridize the Target Probe

1. Remove 1X PBS, and add 3 drops of pre-mixed Target Probe or Control Probe to each well.
2. Cover the 96-well plate with lid and incubate plate in the HybEZ^™ Oven at 40°C for 2 HRS.
3. Remove 96-well plate from the HybEZ^™ Oven. Decant liquid.
4. Wash cells with 200 µL of 1X Wash Buffer 200 µL per well. Incubate at RT for 2 MIN.
5. Repeat step 5 twice using fresh 1X Wash Buffer each time.

Hybridize Amp 1

1. Remove excess liquid from the plate, and add ~3 drops of RNAscope Multiplex FL v2 Amp 1 to each well. Make sure to completely cover the cells.
2. Insert the plate into the HybEZ^™ Oven for 30 MIN at 40°C.
3. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Make sure each well contains liquid.
4. Repeat with fresh 1X Wash Buffer.

Hybridize Amp 2

1. Remove excess liquid from the plate, and add ~3 drops of RNAscope Multiplex FL v2 Amp 2 to each well. Make sure to completely cover the cells.
2. Insert the plate into the HybEZ^™ Oven for 30 MIN at 40°C.
3. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Make sure each well contains liquid.
4. Repeat with fresh buffer.

Hybridize Amp 3

1. Remove excess liquid from the plate, and add ~3 drops of RNAscope Multiplex FL v2 Amp 3 to each well. Make sure to completely cover the cells.
2. Insert the plate into the HybEZ^™ Oven for 15 MIN at 40°C.

NOTE:  Prepare TSA Plus Fluorophores during this step. See the following section.

1. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Make sure each well contains liquid.
2. Repeat with fresh buffer.

Prepare TSA^™ Plus Fluorophores

1. Determine the volume of TSA^® Plus fluorophore needed (100-120uL uL for each well).
2. Dilute the TSA^® Plus fluorophore stocks using the RNAscope Multiplex TSA buffer provided in the RNAscope Multiplex Fluorescent Kit v2. Follow these recommendations:

*Start with a dilution of 1:1500 and adjust based on signal intensity.

NOTE:  Store diluted TSA^® Plus fluorophores up to one month at 2–8°C in the dark.

NOTE:   You may replace TSA^® Plus fluorophores with certain Opal^™ dyes from Perkin Elmer.

Develop HRP-C1 signal

1. Remove excess liquid from the plate, and add ~3 drops RNAscope Multiplex FL v2 HRP-C1 to each well. Make sure to completely cover the cells.
2. Insert the plate into the HybEZ^™ Oven for 15 MIN at 40°C.
3. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Make sure each well contains liquid.
4. Repeat with fresh 1X Wash Buffer.
5. Remove excess liquid from the plate, and add 100–120 µL diluted TSA^® Plus fluorescein to each well.
6. Incubate for 30 MIN at 40°C.
7. Wash the plate in 1X Wash Buffer for 2 MIN at RT. Repeat with fresh buffer.
8. Remove excess liquid from the plate, and add ~3 drops RNAscope Multiplex FL v2 HRP blocker to each well.
9. Insert the plate into the HybEZ^™ Oven for 15 MIN at 40°C.
10. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Repeat with fresh buffer.

NOTE:  You can mix and match channels and fluorophores. Do not assign the same fluorophore to more than one channel.

Develop HRP- C2 signal

1. Remove excess liquid from the plate, and add ~3 drops RNAscope Multiplex FL v2 HRP-C2 to each well.  Make sure to completely cover the cells.
2. Insert the plate into the HybEZ^™ Oven for 15 MIN at 40°C.
3. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Make sure each well contains liquid.
4. Repeat with fresh 1X Wash Buffer.
5. Remove excess liquid from the plate, and add 100-120µL diluted TSA^® Plus Cyanine 3 to each well,
6. Incubate for 30 MIN at 40°C.
7. Wash the plate in 1X Wash Buffer for 2 MIN at RT. Repeat with fresh buffer.
8. Remove excess liquid from the plate, and add ~3 drops RNAscope Multiplex FL v2 HRP blocker to each well.
9. Insert the plate into the HybEZ^™ Oven for 15 MIN at 40°C.
10. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Repeat with fresh buffer.

NOTE:  You can mix and match channels and fluorophores. Do not assign the same fluorophore to more than one channel.

Develop HRP-C3 signal

1. Remove excess liquid from the plate, and add ~3 drops RNAscope Multiplex FL v2 HRP-C3 to each well. Make sure to completely cover the cells.
2. Insert the plate into the HybEZ^™ Oven for 15 MIN at 40°C.
3. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Make sure each well contains liquid.
4. Repeat with fresh 1X Wash Buffer.
5. Wash the plate in 1X Wash Buffer for 2 MIN at RT. Repeat with fresh buffer.
6. Remove excess liquid from the plate, and add ~3 drops RNAscope Multiplex FL v2 HRP blocker to each well.
7. Insert the plate into the HybEZ^™ Oven for 15 MIN at 40°C.
8. Wash the plate with 1X Wash Buffer for 2 MIN at RT. Repeat with fresh buffer.

NOTE:  You can mix and match channels and fluorophores. Do not assign the same fluorophore to more than one channel.

Counterstain  with DAPI

1. Remove excess liquid from the plate, and add ~2 drops of DAPI to each well.
2. Incubate for 30 SEC at RT.
3. Remove DAPI, and wash with 200 µl/well of 1X PBS.
4. Remove 1X PBS, and replace with fresh 1X PBS.

NOTE:  You may store the plate at 4ºC for up to 48 HRS with an adhesive seal before imaging.

Evaluate the Results

Examine the plate under a fluorescent microscope at 20–40X magnification using appropriate filter sets.

1. Remove excess liquid from the plate, and add 100–120 µL diluted TSA^® Plus Cyanine 5 to each well.
2. Incubate for 30 MIN at 40°C

Obtaining Support

For the latest services and support information, contact our Technical Services.

At the website, you can:

• Access telephone and fax numbers to contact Technical Support and Sales.
• Search through FAQs.
• Submit a question directly to Technical Services.

NOTE:  This protocol is for limited distribution only. The protocol is an early development draft, and has not through ACD’s complete development and verification process.