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Immunofluorescence

ND Nicolas Damond

Last updated date: Mar 17, 2020 Views: 1061 Forks: 0

original An abbreviated version of this protocol was published in eLife in Apr, 2016
Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist
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- Starting material: cryosections, 10um-thick.

- Rehydration: 15 min at RT

- Permeabilization: 20 min at RT in PBS-Triton 0.1%

- Wash 3 x 5 min in PBS

- Blocking: 30 min at RT in blocking buffer (3% BSA, 0.1% Tween in PBS)

- Primary antibodies: 2h at RT

Host
Target
Dilution
Vendor
Guinea pig
Insulin
1:400
DAKO
Mouse
Glucagon
1:250 to 1:1000
Sigma
Rabbit
GFP
1:200
Molecular Probes Inc.

- Wash 3 x 5 min in PBS

- Secondary antibodies: 45 mn at RT

Host
Target
Dilution
Fluorophore
Vendor
Goat
Guinea Pig
1:500
AF568
Molecular Probes Inc.
Goat
Mouse
1:500
AF647
Molecular Probes Inc.
Goat
Rabbit
1:500
AF488
Molecular Probes Inc.
-
DAPI
1:500
-
ThermoFisher

- Wash 3 x 5 min in PBS

- Mount with mounting media and coverslip

Copyright: Content may be subjected to copyright.
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Damond, N(2020). Immunofluorescence. Bio-protocol Preprint. bio-protocol.org/prep249.
  2. Damond, N., Thorel, F., Moyers, J. S., Charron, M. J., Vuguin, P. M., Powers, A. C. and Herrera, P. L.(2016). Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist. eLife. DOI: 10.7554/eLife.13828

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