Mar 20, 2025
Immunofluorescence_250206
- Lucia Grijpink1
- 1Leiden University Medical Center
Protocol Citation: Lucia Grijpink 2025. Immunofluorescence_250206. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr9qzzvmk/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: February 06, 2025
Last Modified: March 20, 2025
Protocol Integer ID: 119682
Keywords: immunofluorescence, IF, IHC, immunohistochemistry
Abstract
Protocol for an immunofluorescence assay on paraffin slices.
Attachments
Materials
- Ethanol
- Xylene
- Citric acid buffer (tri-sodium citrate 2,94 g in 1000 ml distilled water, pH adjusted to 6.0 using 1N HCL)
- Coverslips
- Distilled water
- MilliQ
- DAPI (D3571, Invitrogen)
- Prolong Gold (P36934, Invitrogen)
- PAP pen
- PBS
- Washing buffer: 0.05% Tween-20 (8.22184, Merck Group) in PBS
- Blocking buffer: 1% BSA (A3059-50G, Sigma-Aldrich ) and 5% normal donkey serum (ab7475, Abcam) in washing buffer
Rehydration
Rehydration
- Xylene, 5 min
- Xylene, 5 min
- Xylene, 5 min
- 96% Ethanol, 5 min
- 96% Ethanol, 5 min
- 96% Ethanol, 5 min
- 70% Ethanol, 5 min
- 50% Ethanol, 5 min
- MilliQ/aqua dest, 5 min
- MilliQ/aqua dest, 5 min
- MilliQ/aqua dest, 5 min
Antigen retrieval
Antigen retrieval
- Add citric buffer in microwaveable vessel, use sufficient volume to cover the slides by at least a few cm
- Place vessel in microwave
- Warm up until boiling
- Place slides in
microwavable vessel
- Place the vessel back in the microwave for 12 min. Use non-sealed vessel to allow for evaporation. Watch out for boiling over
- After 12 min, remove the vessel and run cold tap water into it for 10 min
Staining
Staining
- Encircle tissue with PAP pen
- Wash for 5 minutes with Washing buffer
- Block for 30 minutes with Blocking buffer
- Incubate with primary Ab in Blocking buffer overnight at 4°C in a humidified chamber
- Wash 3 times with Washing buffer, 5 minutes each
- Block for 10 minutes with Blocking buffer
- Incubate with secondary Ab in Blocking buffer at RT for 60 minutes (in the dark) including 1:1000 DAPI
- Wash 3 times with Washing buffer, 5 minutes each
- Mount cells in Prolong gold and affix coverslip