Jul 22, 2020
Marmoset NHP Free Floating Anti-GFP Antibody Stain After HCR FISH
This protocol is a draft, published without a DOI.
- 1Massachusetts Institute of Technology
Protocol Citation: Guoping Feng Lab 2020. Marmoset NHP Free Floating Anti-GFP Antibody Stain After HCR FISH. protocols.io https://protocols.io/view/marmoset-nhp-free-floating-anti-gfp-antibody-stain-bij9kcr6
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: In development
We are still developing and optimizing this protocol
Created: July 14, 2020
Last Modified: July 22, 2020
Protocol Integer ID: 39265
Abstract
A protocol for staining NHP tissue for anti-GFP antibody after staining for markers of intrest with HCR FISH.
Materials
MATERIALS
Goat serumSigma AldrichCatalog #G9023
GFP Polyclonal AntibodyInvitrogen - Thermo FisherCatalog #A-11122
Goat anti-Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488Invitrogen - Thermo FisherCatalog #A-11034
Primary Antibody
Primary Antibody
3d
3d
Permeabilized marmoset brain sections (100 µm , fixed previously in 4 % volume PFA) in 1.2 % volume Triton X-100 in PBS for 00:30:00 .
Block sections with 10 % volume NS (natural serum depending on Secondary Antibody) and PBT [0.1 % volume Triton X-100, 1X PBS] for 01:00:00 .
Incubate primary antibody (1:500 whole mount - .002 % volume ) with 3 % volume NS and PBT-Azide [0.2 % volume NaN3, 1X PBT] at 4 °C for 72:00:00 .
Secondary Antibody
Secondary Antibody
2d
2d
Wash sections in 1X PBS for 00:10:00 6X.
Incubate with secondary antibody (1:500 whole mount - .002 % volume ) in PBT at 4 °C for 24:00:00 .
Wash sections in wash buffer for 00:10:00 6X.
Stain for DAPI (1:10000 in PBS - .0001 % volume ).
Wash sections in 1X PBS for 00:05:00 2X.