Sep 12, 2024

Public workspaceManual Basescope Duplex Assay

DOI
dx.doi.org/10.17504/protocols.io.x54v9245ml3e/v1
  • 1University of Minnesota
  • Team Lee
Jane Balster
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DOI: dx.doi.org/10.17504/protocols.io.x54v9245ml3e/v1
Protocol Citation: Hector Martell Martinez 2024. Manual Basescope Duplex Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9245ml3e/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: August 21, 2024
Last Modified: September 12, 2024
Protocol Integer ID: 107522
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 000592
Abstract
This protocol details the manual basescope duplex assay.
Materials
  • ReagentBaseScope™ Duplex Reagent KitAdvanced Cell DiagnosticsCatalog #323800
Assign C1 green probe to morphology.

DAY 1

  • Fresh Xylene
  • 100% EtOH
  • Hydrogen peroxide, Protease III
  • Target Retrieval
  • Amount25 mL 10X Target Retrieval + Amount225 mL Nanopure Water (TV = Amount250 mL )
  • ReagentImmEdge Hydrophobic Barrier PenAdvanced Cell DiagnosticsCatalog #310018
  • Nanopure water
  • Probes (Green/C1 Morphology, Red/C2 Ercc1)
  • 1X Wash Buffer
o Warm 50X Buffer to Temperature40 °C (Duration00:10:00 -Duration00:20:00 )
o Amount40 mL 50X Buffer + Amount1960 mL Nanopure Water (TV=2 L)
  • SSC
o Amount60 mL 20X SSC + Amount180 mL Nanopure Water (TV = Amount240 mL )

DAY 2

  • AMP1 – AMP12
  • Wash Buffer
  • Red-B
  • Red-A
  • Green-B
  • Green-A
  • Hematoxylin (MHS16)
o Amount100 mL Gill’s Hemotoxylin + Amount100 mL Nanopure water (TV= Amount200 mL )
  • ReagentVector Labs Vectamount (60mL)Advanced Cell DiagnosticsCatalog #321584
  • 0.02% ammonia water
o Amount143 µL 28% ammonium hydroxide + Amount200 mL Nanopure Water (TV = Amount200 mL )
  • Fresh Xylene














Day 1
Day 1
Bake slides @ Temperature60 °C for Duration01:00:00 .

1h
Temperature
After baking, wet humidifying paper, warm oven and slide tray to Temperature40 °C .
Temperature
Deparaffinize
Xylene – Duration00:05:00 w/ agitation.

  • Meanwhile… turn steamer on, make 1X Target Retrieval solution, turn slide warmer to Temperature60 °C .

5m
Temperature
Fresh Xylene – Duration00:05:00 w/ agitation.
5m
100% EtOH – Duration00:02:00 w/ agitation.
2m
Fresh 100% EtOH – Duration00:02:00 w/ agitation.
2m
Dry on slide warmer – Temperature60 °C for Duration00:05:00 .

5m
Temperature
Hydrogen Peroxide
Cover tissue with hydrogen peroxide and incubate covered for Duration00:10:00 .

  • Meanwhile… Boil 1X Target Retrieval solution in microwave then place in steamer.

10m
Incubation
Tap solution off on absorbent paper.
Wash in nanopure water (up and down 3-5 times then incubate for 2 min) 2X.

  1. Wash in nanopure water (up and down 3-5 times then incubate for Duration00:02:00 ) (1/2).
  2. Wash in nanopure water (up and down 3-5 times then incubate for Duration00:02:00 ) (2/2).

4m
Incubation
Wash
Target Retrieval
Transfer samples to Temperature99 °C 1X Target Retrieval solution for Duration00:30:00 .

30m
Temperature
Wash in nanopure water (up and down 3-5 times then incubate for ~Duration00:00:15 )

15s
Incubation
Wash
Transfer to second container of 100% EtOH in fume hood for Duration00:03:00 .

3m
Dry on slide warmer – Temperature60 °C for Duration00:05:00 .

5m
Temperature
Hydrophobic Barrier
Create barrier with hydrophobic pen – let dry for Duration00:05:00 @ TemperatureRoom temperature .

  • Possible stopping point: Dry O/N @ TemperatureRoom temperature .

5m
Temperature
Protease III
Cover tissue with Protease III for mouse or cell pellets. Protease IV for human tissues.
Incubate for Duration00:30:00 @ Temperature40 °C .

  • Meanwhile… Warm probes to Temperature40 °C inside oven for ~Duration00:10:00 .

40m
Incubation
Temperature
Wash in nanopure water w/ slight agitation 2X.
Wash
Hybridize Probes
Cover tissue (~Amount120 µL ) with 1X probe mix (see below).

  1. C1 probes are 1X; C2 probes are 50X.

  • Dilute C2 probe using C1 probes to 1X.

Incubate for Duration02:00:00 @Temperature40 °C .

2h
Incubation
Temperature
Wash
Wash
Wash in 1X Wash buffer for Duration00:02:00 2X.

  • Store O/N in 5X SSC @ TemperatureRoom temperature .

2m
Wash
Temperature
Day 2
Day 2
4h 6m
4h 6m
Turn on oven to Temperature40 °C . Spray everything with RNase Away.

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).

4m
Wash
Temperature
Cover tissue with AMP1 – incubate for Duration00:30:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).

34m
Incubation
Wash
Temperature
Cover tissue with AMP2 – incubate for Duration00:30:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
34m
Incubation
Wash
Temperature
Cover tissue with AMP3 – incubate for Duration00:15:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
19m
Incubation
Wash
Temperature
Cover tissue with AMP4 – incubate for Duration00:30:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
34m
Incubation
Wash
Temperature
Cover tissue with AMP5 – incubate for Duration00:30:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
34m
Incubation
Wash
Temperature
Cover tissue with AMP6 – incubate for Duration00:15:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
19m
Incubation
Wash
Temperature
AMP 7 & 8 are @ TemperatureRoom temperature

Cover tissue with AMP7 – incubate for Duration00:30:00 @ TemperatureRoom temperature .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
34m
Incubation
Wash
Temperature
Cover tissue with AMP8 – incubate for Duration00:15:00 @ TemperatureRoom temperature .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
19m
Incubation
Mix
Temperature
Detect Red signal
Make enough solution for each sample.

Amount2 µL Red-B + Amount120 µL Red-A = 1 sample (Light sensitive).

Cover tissue with solution – incubate for Duration00:10:00 @ TemperatureRoom temperature .

10m
Incubation
Temperature
  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
4m
Wash
AMP 9 & 10 @ Temperature40 °C .

Cover tissue with AMP9 – incubate for Duration00:15:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).

19m
Incubation
Wash
Temperature
Cover tissue with AMP10 – incubate for Duration00:15:00 @ Temperature40 °C .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).

Turn off Oven!!!! Rest of protocol is at TemperatureRoom temperature .

19m
Incubation
Wash
Temperature
Cover tissue with AMP11 – incubate for Duration00:30:00 @ TemperatureRoom temperature .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
34m
Incubation
Wash
Temperature
Cover tissue with AMP12 – incubate for Duration00:15:00 @ TemperatureRoom temperature .

  • Wash slides in 1X Wash buffer for 2 min 2X.

  1. Wash slides in 1X Wash buffer for Duration00:02:00 (1/2).
  2. Wash slides in 1X Wash buffer for Duration00:02:00 (2/2).
19m
Incubation
Wash
Temperature
Detect Green Signal
Make enough solution for each sample.

  • Amount2.4 µL Green-B + Amount120 µL Green-A = 1 sample (Light sensitive).

Cover tissue with solution – incubate for Duration00:10:00 @ TemperatureRoom temperature .

10m
Incubation
Temperature
Wash slides in 1X Wash buffer for Duration00:05:00 .

5m
Wash
Rinse slides in nanopure water.
Wash
Counterstain: if hematoxylin is too dark, it will mask signal; especially green).
Incubate slides in 50% hematoxylin staining solution for Duration00:00:30 – Duration00:01:00 .

  • Slides should turn purple.
1m 30s
Incubation
Take out immediately and wash in nanopure water 3-5X.

  • Until slides are clear.
Wash
Dip slides up and down 2-3X in 0.02% ammonia water (should turn blue).
Wash in nanopure water 3-5X.
Wash
Dry Slides.
Dry for Duration00:15:00 @ Temperature60 °C .

15m
Temperature
Mounting.
Dip quickly in FRESH xylene
Place 1-2 drops of VectaMount on the slide.
Coverslip.
Air-dry.
Protocol references
Refer to ACD manual Basescope duplex assay for reference.