Dual In Situ Hybridization/Immunofluorescence

Bake slides in a dry oven for 1h 0m 0s at 60°C. Use slides within a week.

De-paraffinize slides in fresh xylenes, then in 100% ethanol.

De-paraffinize slides for 0h 5m 0s in fresh xylenes. (1/4)

De-paraffinize slides for 0h 5m 0s in fresh xylenes. (2/4)

De-paraffinize slides for 0h 2m 0sin 100% ethanol. (3/4)

De-paraffinize slides for 0h 2m 0sin 100% ethanol. (4/4)

Place slides on absorbent paper and dry in the oven from0h 5m 0s at 60°C or until dry.

Place slide horizontally in an incubation tray. Add ~5-8 drops of RNAscope Hydrogen Peroxide to cover each section. Incubate for 0h 10m 0s at 60Room temperature.

Dab solution off and move to a rack in distilled water. Move up and down 5 times. Repeat with a fresh boat of distilled water.

Dilute Target Retrieval Regents (RNAscope) 1:10 in dH₂O (25mL/225mL dH2O/boat). Mix well.

Place in microwave for 0h 15m 0sat 95°C.

Transfer slides to a slide boat with 200mL distilled water for 0h 0m 15s.

Transfer the slides to 100% ethanol for 0h 3m 0s.

Dry the slides in a 60°C incubator (or 60Room temperature) for 0h 5m 0s.

Draw a hydrophobic barrier onto slides with ImmEdge pen. Do NOT due for fluorescent slides. Let the barrier dry for0h 5m 0s. OPTIONAL PAUSE POINT 0h 5m 0s at60Room temperature.

Place a wet Humidifying Paper in an incubation tray and warm for 0h 30m 0s at 40°C(TC incubator). Keep the tray in the incubator when not in use. Insert the slides into the incubation tray.

Add ~5 drops RNAScope Protease Plus (Protease III-Cheadle) to cover each section and place tray into the incubator at40°C for0h 30m 0s (standard) (0h 15m 0s-Otero-Garcia).

Wash slides with 200 mL+ distilled water and slight agitation.

Wash slides with 200mL+ distilled water and slight agitation. (1/2)

Wash slides with 200mL+ distilled water and slight agitation. (2/2)

Wash Buffer : Warm 50x Buffer to 40°C for 0h 10m 0s to0h 20m 0s. Add 980mL distilled water to 20mL of RNAscope Wash Buffer in a 1Lbottle. May need 1L to 2L per run. Mix well. Can be stored for up to one month.

Probes : Prepare only those probes needed.

Warm probes for 0h 10m 0s at 40°C, then let cool to40Room temperature. Add 1 volume C2 and volume C3 probes to 50 volumes C1 probe in a tube (e.g. 200µL C1 + 4µL C2). Invert to mix. Store at 4°C for up to 6 months.

Reagents : Warm AMP1-3, HRP-C1-3 and HRP blocks at 4Room temperature.

(Optional) Saline Sodium Citrate : 175.3g NaCl + 88.2g sodium citrate in 800mListilled water. Adjust to7.0 with 1Molarity (M) HCl. Add water to a final volume of 1L. Sterilize by autoclaving and store at 4Room temperature for up to 2 months.

Remove liquid from slides. Add 4-6 drops (6 drops=180µL) of the probe mix to slides. Incubate in incubator for 2h 0m 0s at 40°C.

Wash slides with Wash Buffer.

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (1/2)

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (2/2)

OPTIONAL PAUSE POINT : Store slides in 5x SSC 0h 2m 0s at 40Room temperature.

Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 Amp 1 to each slide. Incubate in incubator for 0h 30m 0s at40°C.

Wash slides with Wash Buffer.

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (1/2)

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (2/2)

Repeat steps 22 and 23 for Amp 2 and Amp 3. Amp 3 only requires 0h 15m 0s at 40°C.

During this incubation, dilute necessary Opal Dye fluorophores in TSA Buffer (1:1500 standard).

Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 HRP-C1 to each slide. Incubate in incubator for 0h 15m 0s at 40°C.

Wash slides with Wash Buffer.

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (1/2)

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (2/2)

Remove liquid from slides. Add 200µL Opal 520 to each slide. Incubate in HybEZ Oven for0h 30m 0sat 40°C.

Wash slides with Wash Buffer.

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (1/2)

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (2/2)

Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 HRP Blocker to each slide. Incubate in incubator for 0h 15m 0s at 40°C.

Wash slides with Wash Buffer.

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (1/2)

Wash slides with Wash Buffer 0h 2m 0s at 40Room temperature. (2/2)

STOP HERE IF USING JUST C1 PROBE. Continue to Immunofluorescence .

Repeat steps 26-32 with HRP-C2 and Opal 570, and again with HRP-C3 and Opal 690.

4% PFA fix for 0h 15m 0s at 4°C.

Then, wash with PBS-Otero-Garcia for 0h 4m 0s. (1/2)

Wash with PBS-Otero-Garcia for 0h 4m 0s. (2/2)

Wash in0.1Molarity (M) Tris buffer, 7.6 0h 5m 0s. Discard all Tris washes.

Block in 0.1Molarity (M) Tris/2% FBS (Tris/FBS) 0h 30m 0s+. Keep blocking solution for up to 2 weeks @ 4°C.

Dilute primary antibodies in Tris/FBS), and prepare humidified chamber(s) by soaking towel in the middle of the slide chamber(s).

Wipe excess fluid off back of slides and from around tissue and apply200µL of primary antibody to slides.

Incubate at4°C in humidified chamber0h 45m 0s to 2h 0m 0s at 4Room temperatureor 2h 0m 0s at 4°C.

Rinse off antibody from tissue using Tris.

Wash in Tris 0h 5m 0s.

Block in Tris/FBS 0h 5m 0s.

Dilute fluorophore-conjugated secondary antibody 1:500 in Tris/FBS and apply 200µL to wiped slides. Incubate at4Room temperature for 2h 0m 0s or 2h 0m 0sat 4°C.

Rinse off slides with Tris.

Wash in running tap H₂O for 0h 5m 0s.

Wash in Tris for 0h 5m 0s in green boats.

Coverslip using non-photobleaching reagent (Prolong Gold with DAPI or FluorMount with DAPI). Allow to dry completely before imaging on scanner.