May 14, 2024
Fixation and Immunostaining protocol
- 1University College of London;
- 2NYSCF
Protocol Citation: gustavo.parfitt Parfitt, Gist Croft 2024. Fixation and Immunostaining protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8wowl5r/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: May 14, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 99797
Keywords: ASAPCRN
Abstract
Fixation and Immunostaining protocol for tissue and cell culture
Materials
Buffers
2x Fix: 8% PFA in 2xPBS: Prepare from
32%PFA in sealed EMS ampule, + 8ml 10x PBS, + 22ml ddH20, stable light
protected at 4 degrees for 2 weeks.
Fix: prepare fresh on day of use. Mix 2x Fix with ddH20, 1:1
Quench: Wash Buffer + 100mM Glycine + 0.1%
Sodium Azide
Block: Wash Buffer + 10% normal donkey
serum + 0.1% Sodium Azide
Wash: 1x PBS + 0.1% Triton-X 100
DAPI 50x stock: dilute -80 frozen DAPI
aliquot (50Kx concentrate) 1:1000 in
filtered TC grade H2O; store LP, 4°C up to 4 months
Prepare 1x Fix by dilution from 2x Fix (see Description)
Gently aspirate culture supernatant, wash gently with PBS, and replace with cold Fix buffer
Incubate 30min @ 4° C or on ice
30m
Aspirate fix and wash gently 3x with PBS (incubate 1-2min each)
Aspirate and add Quench to permeabilize cells/tissue, at least 15 min room temperature (RT). Note: this and all steps in humidified chamber (HC). Can store samples for weeks/months at 4 degrees
Aspirate and add Block buffer, 30 min RT
30m
Incubate with primary antibodies cocktail (1-2 hrs RT or 4°C overnight (ON)
Wash 3x 5min (wash buffer)
15m
Prepare secondary antibodies in Wash buffer (1:500-1:1000), filter, light protect (LP).Note: This and all subsequent steps LP
17h
Incubate with secondary antibodies 1hr RT, light protected (LP)
1h
Wash 3x 5min
15m
Incubate with 1x DAPI in Wash buffer 5-10 min
10m
Wash 1x
Rinse very briefly with filtered H2O, 2x
Remove excess water, do not dry samples
Add Fluoromount-G (~20ul/well or ~100ul/slide) and apply No 1.5H coverslip (slowly lower, no bubbles) or other mountant
Dry ON, LP, RT