Sep 12, 2022
Immunochemistry on paraffin sections
- 1Vall d'Hebron Research Institute
Protocol Citation: Marta Gonzalez-Sepulveda, Joan Compte, Miquel Vila 2022. Immunochemistry on paraffin sections. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9my4g8j/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 12, 2022
Last Modified: November 10, 2023
Protocol Integer ID: 69880
Abstract
Immunochemistry 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 Xilen.
Wash 1x10 min in ethanol 100%.
Wash 1x10 min in ethanol 95%.
Wash 1x5 min in ethanol 70%.
Wash 2x5 min in TBS 1X.
2. Blocking endogenous peroxidase:
2. Blocking endogenous peroxidase:
Put the slides 10 min in endogenous peroxidase blocking solution: TBS 1x + 3% H2O2 + 10% methanol
Wahsing
Wahsing
Wash 3x5 min in buffer TBS 1X.
Antigen retrieval
Antigen retrieval
Put sections in 200 mL of citrate buffer 10mM, pH 6
Put sections in a boiling water bath for 20 min
Let sections cool down for 20 min at RT
Washing
Washing
Wash 3x5 min in TBS 1X.
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
Blocking in TBS 1X + 5% NGS (200uL/slides) 1h at RT
1ary antibody
1ary antibody
Gently wipe off the water ofthe slides
Put 200ul/slide of TBS 1X + 2% NGS + primary Ab48/72h (it depends of the Ab) at 4ºC (cold room)
Washing
Washing
Wash 3x5min in TBS 1X Buffer.
2ary antibody
2ary antibody
Gently wipe off the water ofthe slides
Put 200ul/slide TBS 1X + 2% NGS + Secondary Ab 1h at RT.
At this step it is important to prepare ABC solution and let it, at least, 30 min on the shaker
Washing
Washing
3x5min in TBS 1X
Peroxidase staining
Peroxidase staining
Incubate 1 hour at RT with ABC solution (4drops A/B in 10 mL of TBS 1x)
Washing
Washing
3x5min in TBS 1X
Developing preparation
Developing preparation
In aluminium foil: DAB Standard Kit: 1 drop of reagent B in 1 mL of reagent A (gives rise to brown staining)
In aluminium foil: Vector SG: 3 drops Chromogen + 3 drops Hydrogen Peroxide in 5mL PBS (gives rise to blue staining)
Developing
Developing
Cover tray plates with aluminium foil
Put 200 uL on each section and put a cardboard box on it to keep darkness for a time ranging from 3-15 minutes depending on the antibody used
Remove with an air-pump equipment and clean the material with bleach
Washing
Washing
3x5min in TBS 1X
Dehydratation
Dehydratation
1 min in ethanol 70%
1 min in ethanol 95%
1 min in ethanol 100%.
2x5 min in xylene
Mounting
Mounting
Puta line of mounting medium(DPX) byslide.Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles
Let dry the slides on the tray in the hood overnight