Feb 03, 2025
Immunofluorescence staining and image analysis
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Immunofluorescence staining and image analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr3k7g5b/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: November 06, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 111683
Funders Acknowledgements:
ASAP
Abstract
Immunofluorescence staining and image analysis
2D cultures IF
2D cultures IF
Seed cells on coverslips and fix with 4% paraformaldehyde (PFA) for 10 minutes
Rinse cells with phosphate-buffered saline (PBS)
Block cells with 10% normal goat or donkey serum (NGS/NDS) in PBS containing 0.1% Triton X-100 (PBST) at room temperature for 1 hour
Incubate cells overnight at 4°C with primary antibodies diluted in PBST containing 5% NGS/NDS
The next day, after washing with PBS 3 times for 5', incubate cells for 2 hours at room temperature with Alexa 488/568/647-conjugated secondary antibodies at a 1:1000 dilution, diluted in PBST containing 5% NGS/NDS
Remove secondary antibodies and stain nuclei with 1 µM DAPI for 5 minutes
Wash coverslips 3 times for 10'
Mount coverslips on glass slides with Dako mounting medium
Brain organoids and Assemblods IF
Brain organoids and Assemblods IF
For brain organoids and assembloids, fix samples with 4% (w/v) PFA for 1 hour
Equilibrate organoids in 30% sucrose in PBS overnight at 4°C
Embed organoids in OCT compound the following day
Cryosection organoids to 20 µm thickness and mount on SuperFrost Plus slides
Block sections with 10% NGS in PBS containing 0.5% Triton X-100
Incubate sections with primary antibodies diluted in PBST containing 5% NGS/NDS overnight
After washing sections with PBS 3 times for 5' Incubate with secondary antibodies diluted in PBST containing 5% NGS/NDS for 3 hours
(If BODIPY staining is needed, incubate cells or tissues with 1 µM BODIPY 493/503 dye in PBS for 30 minutes at room temperature)
(For protein aggregate detection, stain sections with 10 µM thioflavin-T for 30 minutes at room temperature)
Stain sections with 1 µM DAPI for 5 minutes
Wash sections three times with PBS for 10'
Mount sections with Dako mounting medium
Mouse tissue IF
Mouse tissue IF
For mouse brain tissue, perform antigen retrieval on free-floating brain slices for 20 minutes in citrate buffer at 80°C
Block tissues with mouse-on-mouse (MOM) blocking reagent following manufacturer’s instructions
Incubate with primary antibodies overnight using MOM protein concentrate
After washing in PBS 3 times 5', incubate with secondary antibodies for 3 hours
Stain sections with 1 µM DAPI for 5 minutes
Wash sections three times with PBS for 10'
Mount sections with Dako mounting medium
Image analysis
Image analysis
Acquire images using a Leica TCS SP8 confocal microscope with a 60×/1.4 numerical aperture objective or an EVOS M7000 Imaging System with a 20×/0.4 numerical aperture
For quantification, analyze p21-, p16-, and γH2AX-positive nuclei in CellProfiler with DAPI as a counterstain
Calculate fluorescence intensity per area using Fiji