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Whole-mount Xenopus Immunostaining Protocol

AW Andrea Elizabeth Wills

Last updated date: Nov 11, 2020 Views: 1010 Forks: 0

original An abbreviated version of this protocol was published in eLife in Apr, 2020
Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors
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Whole Mount Xenopus Immunostaining Protocol

Adapted from Helen Willsey, modified after Peter Walentek and Susanne Bogusch

 

Reagents:

-1x MEM with 3.7% formaldehyde

-CAS-Block (Invitrogen #00-8120)

-1X PBS

-1X PBT (PBS + 0.1% Triton X-100)

-1X bleach: per ml= 50uL Formamide, 40uL Peroxide, 910uL PBS

 

Protocol:

1.     Fix tadpoles in 1x MEM with 3.7% formaldehyde 30min-1hr RT. (NO LONGER THAN 1hr)

2.     Optional: bleach tadpoles for 2hr RT under light box.

3.     Permeabilize by washing 3x20’ RT PBT.

4.     Block in 10% CAS-Block in PBT 1hr RT.

5.     Incubate in 1o antibody diluted in 100% CAS-Block O/N 4oC.

6.     Wash 3x10’ PBT RT.

7.     Block again in 10% CAS-Block in PBT 30’.

8.     Incubate in 2o antibody diluted in 100% CAS-Block 2hr RT.

9.     Wash 3x10’ PBT RT.

10.  Optional: Incubate in Phalloidin-647 diluted 1:40 in PBT 30’ RT.

11.  Wash 3x20’ PBS.

12.  Mount with Vectashield + DAPI.

 

 

 


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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Wills, A(2020). Whole-mount Xenopus Immunostaining Protocol. Bio-protocol Preprint. bio-protocol.org/prep620.
  2. Kakebeen, A. D., Chitsazan, A. D., Williams, M. C., Saunders, L. M. and Wills, A. E.(2020). Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors. eLife. DOI: 10.7554/eLife.52648

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