Oct 17, 2023
Immunohistochemistry on free-floating cryosections
Forked from Immunochemistry on paraffin sections
- 1Vall d'Hebron Research Institute
Protocol Citation: Miquel Vila 2023. Immunohistochemistry on free-floating cryosections. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx7x8gx1/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: October 17, 2023
Last Modified: November 20, 2023
Protocol Integer ID: 89426
Abstract
Immunochemistry protocol on rodent brain cryosections
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. Section selection
1. Section selection
Collect the cryosections needed for a caudo-rostral representation of each brain region (every fourth section or every sixth section dependeing on the section thinknes, brain region and animal species) into 24-well-plate (3-4 sections per well).
Wash 3x5 min in TBS 1X. Put 500ul per well and aspirate the liquid with an air-pump equipment.
2. Blocking endogenous peroxidase
2. Blocking endogenous peroxidase
Incubate sections in endogenous peroxidase blocking solution: TBS 1x + 3% H2O2 + 10% methanol for 10 min (500uL/well)
Wash 3x5 min in TBS 1X.
3. Blocking
3. Blocking
Incubate sections in blocking in TBS 1X + 5% NGS or NDS (500uL/well) 1h at RT
4. 1ary antibody
4. 1ary antibody
Incubate sections inTBS 1X + 2% NGS or NDS + primary Ab 24/72h (it depends of the Ab) at 4ºC (cold room).
Wash 3x5min in TBS 1X.
5. 2ary antibody
5. 2ary antibody
Incubate sections in 2% NGS or NDS + 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
Wash 3x5min in TBS 1X.
6. ABC incubation
6. ABC incubation
Incubate 1 hour at RT with ABC solution (Ultra-Sensitive or Standard ABC Peroxidase Standard Staining Kit).
Wash 3x5min in TBS 1X.
7. Developing
7. Developing
In aluminium foil: DAB Standard Kit (1 drop of reagent B in 1 mL of reagent A, gives rise to brown staining), or Vector SG (3 drops Chromogen + 3 drops Hydrogen Peroxide in 5mL PBS, gives rise to blue staining).
Put 500 uL on each well 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.
Wash 3x5min in TBS 1X.
8. Mount sections
8. Mount sections
Mount sections into slides and let it dry overnight.
9. Dehydratation
9. Dehydratation
Incubate slides in consecutive ethanol solutions (1 min in ethanol 70%-1 min in ethanol 95%-1 min in ethanol 100%).
Incubate slides in 2x5 min in Xylene.
10. Mount coverslips
10. Mount coverslips
Put a line of mounting medium (DPX) by slide. Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles.
Let dry the slides in the hood overnight.