Jul 04, 2025
Double immunofluorescence – Paraffin-embedded sections
- Raheleh Roudi1
- 1University of Stanford
- Raheleh
Protocol Citation: Raheleh Roudi 2025. Double immunofluorescence – Paraffin-embedded sections. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlky5b6g5r/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: July 04, 2025
Last Modified: July 04, 2025
Protocol Integer ID: 221773
Keywords: double immunofluorescence procedure, double immunofluorescence, different antigens in the same sample, different antigen, primary antibody, different species, paraffin, same sample, antibody
Funders Acknowledgements:
Raheleh Roudi
Grant ID: U24CA264298
Heike E. Daldrup-Link
Grant ID: U24CA264298
Abstract
In order to be able to examine the co-distribution of two (or more) different antigens in the same sample, a double
immunofluorescence procedure can be carried out. Primary antibodies raised in different species can be used either in
parallel (in a mixture) or in a sequential way.
Troubleshooting
Preparation of slides and samples
1.1. Deparaffinize sections in xylene 2x5 min.
1.2. Hydrate with 100% ethanol 2x3 min.
1.3. Hydrate with 95% ethanol 1 min.
1.4. Rinse in distilled water
2. Fixation
2.1 Fix the samples either in ice-cold methanol, acetone (1-10 min) or in 3-4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature.
2.2 Wash the samples twice with ice cold PBS.
3. Permeabilization
3.1 Incubate the samples for 10 min with PBS containing 0.25% Triton X-100
3.2 Wash cells in PBS three times for 5 mins.
4. Blocking and simultaneous incubation
4.1. Incubate tissues with 1% BSA in PBST for 30 min to block unspecific binding of the antibodies
4.2. Incubate tissues in the mixture of two primary antibodies in 1% BSA in PBST in a humidified chamber for overnight at 4o C.
4.3. Decant the mixture solution and wash the cells three times in PBS, 5 mins each wash.
4.4. Incubate cells with the mixture of two secondary antibodies which are raised in different species in 1% BSA for 1 hr at room temperature in dark.
4.5. Decant the mixture of the secondary antibody solution and wash three times with PBS for 5 min each in dark.
5. Counter staining
5.1. Rinse with PBS.
5.2 Incubate tissues on 0.1-1 µg/ml DAPI for 1 min.
6. Mounting
6.1. Seal coverslip with nail polish to prevent drying and movement under microscope.
6.2. Store in dark at -20°C or 4°C.