Apr 05, 2024
BaseScope In Situ Hybridization
- 1Van Andel Research Institute
Protocol Citation: madalynn.erb Erb 2024. BaseScope In Situ Hybridization. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo364zv4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94340
Keywords: ASAPCRN
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
BaseScope in situ hybridization on mouse brain sections
Day 1
Day 1
Use 35μM floating brain sections
Wash sections 3 times (5 minutes) in PBS to remove cryoprotectant solution
Mount tissue onto Superfrost plus slides
Dry slides at room temp and then dip in mQ H2O to remove salt
Dry slides at 60°C for 2 hours
Let tissue dry at room temp overnight – make sure tissue is completely stuck to slides
Day 2
Day 2
Dry slides at 60°C for 30 minutes
Dehydrate slides in 50%, 70%, 100% EtOH, for 5 min each at room temp
Note
Use sterile distilled H2O for the rest of the protocol
Let slides air dry for 5 min at room temp.
Make 200mL Target Retrieval Reagent (1:10 180mL H2O + 20mL 10X Target Retrieval Reagent)
On the bench – add 5-8 drops of Hydrogen Peroxide to tissue sections
Incubate at room temp for 10 min
Rinse slides 2 times in H2O
Submerge in dish of H2O and move slides up and down 3-5 times for each wash
In the steamer heat one dish of H2O and one dish of Target Retrieval Reagent
Check temp of Target retrieval reagent (must be at least 95°C)
After preheating – place slides into H2O for 10 sec to acclimate.
Move slides to Target Retrieval Reagent and steam for 5 min
Remove the water container and the container with slides from the steamer and place on the bench.
Let slides cool on the bench for 20 -30 minutes (wait until temperature of water container has dropped to 30°C)
Wash slides in H2O for 15 sec at room temp
Wash slides in 100% EtOH for 3 min at room temp
Dry slides at 60°C for ≥5 min
Use an Immedge hydrophobic barrier pen to draw a barrier around your tissue section(s)
Let the barrier dry ≥ 1 min or overnight at room temp (you can take a break at this point)
Turn on RNA Scope oven – set to 40°C
Wet humidifying paper (filter paper) completely with H2O and place in the Humidity Control Tray
Warm the covered tray in the oven for 30 minutes before use
Place slides in slide rack and add ~5 drops of Protease IV
Incubate in pre-warmed RNAscope oven in the humidity tray at 40°C for 30 min
Wash slides in H2O at room temp
Prepare 1L of wash buffer (20mL of 50X Wash buffer + 980mL H2O)
Heat 50X wash buffer to 40°C for 10-20 min before making 1X wash buffer
Equilibrate each BaseScope reagent and probe to room temp for 30 min before use
Important: Do not let sections dry out between incubation steps
All incubation steps are in the slide rack – remove slides from slide rack to wash between incubations
For the incubations in the HybEZ oven – cover the slide rack with the lid and make sure to lock the oven so that the water in the humidity chamber does not evaporate.
Add ~4 drops of BaseScope probe to completely cover tissue
Incubate in the oven at 40°C for 2 hours
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Add ~4 drops of AMP1 to completely cover tissue
Incubate in the oven at 40°C for 30 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Add ~4 drops of AMP2 to completely cover tissue
Incubate in the oven at 40°C for 30 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Add ~4 drops of AMP3 to completely cover tissue
Incubate in the oven at 40°C for 15 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Add ~4 drops of AMP4 to completely cover tissue
Incubate in the oven at 40°C for 30 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Add ~4 drops of AMP5 to completely cover tissue
Incubate in the oven at 40°C for 30 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Add ~4 drops of AMP6 to completely cover tissue
Incubate in the oven at 40°C for 15 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
We are done with the oven – but keep using the slide rack for incubations at room temp
Add ~4 drops of AMP7 to completely cover tissue
Incubate at room temp for 15 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Staining intensity can be modified by adjusting the AMP7 incubation time
Add ~4 drops of AMP8 to completely cover tissue
Incubate on the bench at room temp for 15 min
Wash 2 times in Wash Buffer at room temp - 2 min each wash
Prepare BaseScope Fast RED working solution (1:60 ratio)
Spin down BaseScope Fast RED-B before using
2uL RED-B + 120uL RED-A : mix well
Use the Fast Red solution within 5 minutes – do not expose to direct sunlight or UV light
Add ~120µL Fast Red solution to each slide / section
Cover sections on tray to protect from light
Incubate at room temp for 10 min
Rinse 2 times in H2O at room temp
Dry slides at 60°C for ≥ 15 min (until slides are completely dry)
The red substrate is alcohol sensitive. DO NOT dehydrate the slides in alcohol, and make sure your reagents are not contaminated with alcohol.
Place 1-2 drops of VectaMount or Ecomount on the slide and coverslip the tissue sections.
Dry slides at room temp for ≥ 5 min