Feb 07, 2018
ICC/IF Protocol
- 1University of Virginia, Charlottesville
- Boster Bio
Protocol Citation: Cj Xia 2018. ICC/IF Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.mgcc3sw
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 in our group and it is working.
Created: January 05, 2018
Last Modified: March 28, 2018
Protocol Integer ID: 9444
Keywords: ICC, IF, immunocytochemistry, immunofluorescence, FITC, light microscopy, fluorescent dye, cell imaging, immunohistochemistry, IHC, immunostaining
Abstract
Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.
This protocol describes the steps for performing ICC/IF.
Guidelines
Tissue preparation is a key to successful IHC experiments. Since no universal tissue preparation method will be ideal for all sample and tissue types, the protocol given here is intended as a starting point from which the experimenter must optimize as needed. All conditions should be standardized in order to ensure reproducible results. Keep in mind that you must be careful not to allow tissues to dry out at any time.
Materials
STEP MATERIALS
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Aqueous Mounting Medium For IHC, ICCCatalog #AR1018
DAPI SolutionBoster BioCatalog #AR1176
DAPI Counterstaining SolutionBoster BioCatalog #AR1177
Antifade Mounting Medium, Prolong FlourescenceBoster BioCatalog #AR1109
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Aqueous Mounting Medium For IHC, ICCCatalog #AR1018
DAPI SolutionBoster BioCatalog #AR1176
DAPI Counterstaining SolutionBoster BioCatalog #AR1177
Antifade Mounting Medium, Prolong FlourescenceBoster BioCatalog #AR1109
Protocol materials
DAPI Counterstaining SolutionBoster BioCatalog #AR1177
Antifade Mounting Medium, Prolong FlourescenceBoster BioCatalog #AR1109
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Aqueous Mounting Medium For IHC, ICCCatalog #AR1018
DAPI SolutionBoster BioCatalog #AR1176
DAPI SolutionBoster BioCatalog #AR1176
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
DAPI Counterstaining SolutionBoster BioCatalog #AR1177
Antifade Mounting Medium, Prolong FlourescenceBoster BioCatalog #AR1109
Aqueous Mounting Medium For IHC, ICCCatalog #AR1018
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Aqueous Mounting Medium For IHC, ICCCatalog #AR1018
DAPI SolutionBoster BioCatalog #AR1176
DAPI Counterstaining SolutionBoster BioCatalog #AR1177
Antifade Mounting Medium, Prolong FlourescenceBoster BioCatalog #AR1109
Sample Preparation - Cell Climbing Slices
Sample Preparation - Cell Climbing Slices
This fixation procedure using paraformaldehyde and formalin fixatives may cause autofluorescence in the green spectrum. In this case, you may try fluorophores in the (i) red range or (ii) infrared range if you have an infrared detection system.
Place settled coverslip in culture bottle or perforated plate.
Take out coverslip after cell growth has reached 60%.
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Immerse the coverslip (cells face up) in cold acetone or 4% paraformaldehyde (AR1068, Boster Bio) or neutral formalin for 10 to 20 minutes. Remember to close the lid to prevent evaporation.
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Wash the coverslip 3X with PBS.
Put the coverslip on filter paper (cells face up).
Remove the liquid on the coverslip and allow it to dry for 8-10 hours.
To thaw the slice, wash with neutral PBS at room temperature for 10-15 minutes (The cell climbing slice can be stored in gelatin at -20°C for one week).
Inactivation
Inactivation
Mix H2O2 with distilled water (v/v: 1:50).
Immerse frozen section or cell climbing slice in the diluted H2O2 at room temperature for 10 minutes.
Wash the section 3X distilled water (1 minute each).
Antigen Retrieval (Proteolytic Induced Epitope Retrieval: PIER)
Antigen Retrieval (Proteolytic Induced Epitope Retrieval: PIER)
Dry the cell slices with filter paper.
Add compound digestion solution (AR0022, Boster Bio) (e.g. Trypsin solution or other enzymatic antigen retrieval solution) to the slices. We recommend the addition of 0.1% Triton to the samples before the digestion because this reduces surface tension and allows reagents to easily cover the entire sample.
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
Incubate the slices at room temperature for 10 minutes.
Wash with 3X PBS (10 minutes each).
Blocking
Blocking
Add 5% BSA blocking solution or normal goat serum to the PIER-treated samples.
Incubate the samples at 37°C for 30 minutes.
37 °C
Shake off extra liquid and dry the samples with filter paper (No washing required).
Primary Antibody Incubation
Primary Antibody Incubation
Dilute primary antibody with antibody diluent (AR1016, Boster Bio) to the concentration recommended by the antibody manufacturer.
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Add the diluted antibody (Recommended concentration: 0.4 µg to 2 µg) to the samples and incubate at 4°C overnight.
4 °C
Wash the samples 3X with PBS (15 minutes each).
Secondary Antibody Incubation
Secondary Antibody Incubation
Dilute biotinylated secondary antibody with antibody diluent to the concentration recommended by the antibody manufacturer.
Add the diluted antibody to the samples and incubate at 37°C for 30 minutes.
37 °C
Wash the samples 3X with PBS (8 minutes each).
Staining
Staining
Incubate the samples at 37°C for 30 minutes. Remember to avoid light.
37 °C
Wash the samples 2X with PBS (Total 2 hours).
Aqueous Mounting Medium For IHC, ICCCatalog #AR1018
Monitor the staining intensity under a fluorescence microscope.
DAPI SolutionBoster BioCatalog #AR1176
DAPI Counterstaining SolutionBoster BioCatalog #AR1177
Check the staining intensity again under a fluorescence microscope.
For slide storage without significant decay in fluorescence signal, add 20 µL of anti-fade solution (AR1109, Boster Bio) to the sample followed by a cover glass. Avoid bubbles.
Antifade Mounting Medium, Prolong FlourescenceBoster BioCatalog #AR1109