Apr 03, 2024
lmmunostaining of Organoid Sections
This protocol is a draft, published without a DOI.
- George Chen1,
- Daniel H. Geschwind1
- 1UCLA
Protocol Citation: George Chen, Daniel H. Geschwind 2024. lmmunostaining of Organoid Sections. protocols.io https://protocols.io/view/lmmunostaining-of-organoid-sections-dbmu2k6w
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: April 03, 2024
Last Modified: April 03, 2024
Protocol Integer ID: 97684
Abstract
lmmunostaining of Organoid Sections derived from human iPS cells.
Materials
- Blocking Buffer: lxPBS + 5% FBS + 1% BSA+ 0.3% Triton X + 0.1% Azide
- lx PBS
- 24-well plate
- Paint brush
- Incubation Buffer: lxPBS + 1% Triton X
- Primary and secondary antibodies
- Fluoromount with DAPI
Place organoid sections into 1ml of PBS in a 24-well plate (1 section per well)
Aspirate off the PBS gently
Add ~600ul of blocking buffer for 20mins at RT on the rocker
20m
Wash 3x with 500ul of PBS and aspirate off PBS
Add primary AB at working concentration and incubate in Blocking Buffer overnight at 4°C (GFAP-1:500)
Wash 3x with 500ul of PBST, and aspirate it off
Add secondary AB at working concentration and incubate in Blocking Buffer at RT on a rocker for 1 hour, making sure to cover the plate with aluminum foil to prevent bleaching of the secondary AB (1:1000 for both secondaries)
Wash 3x in PBST mount onto a slide in water
DAPI staining is 2ug/ml in PBST for 15 min at RT
15m
Wash 3x with PBST
Once the section is on the slide, aspirate off some of the water and add a drop of Fluoromount with DAPI before placing round coverslip on top, being careful not to cause any bubbles underneath
leave overnight at RT to let dry and covered to prevent bleaching
Store covered at 4C