Sep 13, 2022
Immunofluorescence on paraffin sections
- 1Vall d'Hebron Research Institute
Protocol Citation: Joan Compte, Marta Gonzalez-Sepulveda, Miquel Vila 2022. Immunofluorescence on paraffin sections. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbn62vx1/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: September 13, 2022
Last Modified: November 10, 2023
Protocol Integer ID: 69926
Abstract
Immunofluorescence protocol on paraffin-embedded rat brain sections
Materials
Reagents :
- TBS 10X : Tris base 121.1g + NaCl 90g in 1L H2O.pH 7.4.
- TBS 1X-Triton 0,5%
- Xilen
- Ethanol : 100%, 95%, 70%
- Unmasking buffer epitopes : Citrate solution 10mM pH6.0
- Blocking Buffer : TBS 1X + 5% NGS
- 1st Ab : Diluted in1X PBS +2%NGS
- 2nd Ab : Diluted in1X PBS +2%NGS
- Endogenous peroxidase blocking solution : TBS 1x + 3% H2O2(30%) + 10% methanol
1. Deparaffinization and hydratation :
1. Deparaffinization and hydratation :
Put the slides 30 min in the incubator at 60ºC
Wash 3x3 min in xylene
Wash 1x10 min in ethanol 100%
Wash 1x10 min in ethanol 95%
Wash 1x5 min in ethanol 70%
Wash 2x5 min in PBS 1X
Note
IMPORTANT: The buffer used for immunofluorescence is PBS, not TBS as for immunohistochemistry and the endogenous peroxidase blocking step is not required.
Antigen retrieval
Antigen retrieval
Add 200 mL/section of citrate buffer 10mM, pH 6
Put sections in a boiling water bath for 20 min
Let sections cool down for 20 min at room temperature (RT)
Washing
Washing
Wash 3x5 min in PBS 1X-Triton (0.5%)
Blocking
Blocking
Put wet paper in a black box for immunohistochemistry
Gently dry and circle sections with the hydrophobic pen ImmEdge Vector H 4000 without touching the tissue
Add 200 uL/section of blocking solution (5% NGS (in PBS 1X) (200uL/section) + 0.1% Triton (in PBS 1X)) 1h at RT
1ary antibody
1ary antibody
Gently wipe off the water ofthe slides
Put 200ul/section of 1ary antibody in 2% NGS + 0.1% Triton overnight at 4ºC (cold room)
Washing
Washing
Wash 3x5min in PBS 1X-Triton (0.5%)
2ary antibody
2ary antibody
Gently wipe off the water ofthe slides
Put 200ul/section of 2ary antibody in 2% NGS + 0.1% Triton + DAPI for 1 h at RT
Note
Alexa goat anti-mouse 488 (green color) 1:500 or 1: 1000, Alexa goat anti-rabbit 594 (red color) and 1:5000 DAPI (nuclei staining in blue color)
Washing
Washing
Wash 3x5min in PBS 1X
Mounting
Mounting
Mount sections with fluorescent mounting medium. Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles
Storage
Storage
Let dry the slides on the tray in the hood for 5 minutes and store at 4ºC asap