Feb 07, 2018
Version 2
IHC-P Protocol V.2
- 1University of Virginia, Charlottesville
- Boster Bio
Protocol Citation: Cj Xia 2018. IHC-P Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.mzuc76w
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: February 02, 2018
Last Modified: March 28, 2018
Protocol Integer ID: 10004
Keywords: ihc, immunohistochemistry, immunostaining, ihc-p, paraffin embedded
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 the immunohistochemistry method with paraffin-embedded tissue sections.
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
Poly-L-Lysine Solution, 10X, For 100-200 SlidesBoster BioCatalog #AR0003
3-Aminopropyltriethoxysilane (APTES) 10ml 50X ConcentratedBoster BioCatalog #AR0001
3% Hydrogen Peroxide (H2O2) Solution, Lab GradeBoster BioCatalog #AR1108
Citrate Buffer Powder 2L/Pack, For Heat Antigen RetrievalBoster BioCatalog #AR0024
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Poly-L-Lysine Solution, 10X, For 100-200 SlidesBoster BioCatalog #AR0003
3-Aminopropyltriethoxysilane (APTES) 10ml 50X ConcentratedBoster BioCatalog #AR0001
3% Hydrogen Peroxide (H2O2) Solution, Lab GradeBoster BioCatalog #AR1108
Citrate Buffer Powder 2L/Pack, For Heat Antigen RetrievalBoster BioCatalog #AR0024
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Protocol materials
Poly-L-Lysine Solution, 10X, For 100-200 SlidesBoster BioCatalog #AR0003
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Citrate Buffer Powder 2L/Pack, For Heat Antigen RetrievalBoster BioCatalog #AR0024
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
3% Hydrogen Peroxide (H2O2) Solution, Lab GradeBoster BioCatalog #AR1108
Citrate Buffer Powder 2L/Pack, For Heat Antigen RetrievalBoster BioCatalog #AR0024
3% Hydrogen Peroxide (H2O2) Solution, Lab GradeBoster BioCatalog #AR1108
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
3-Aminopropyltriethoxysilane (APTES) 10ml 50X ConcentratedBoster BioCatalog #AR0001
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
3-Aminopropyltriethoxysilane (APTES) 10ml 50X ConcentratedBoster BioCatalog #AR0001
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Poly-L-Lysine Solution, 10X, For 100-200 SlidesBoster BioCatalog #AR0003
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Poly-L-Lysine Solution, 10X, For 100-200 SlidesBoster BioCatalog #AR0003
3-Aminopropyltriethoxysilane (APTES) 10ml 50X ConcentratedBoster BioCatalog #AR0001
3% Hydrogen Peroxide (H2O2) Solution, Lab GradeBoster BioCatalog #AR1108
Citrate Buffer Powder 2L/Pack, For Heat Antigen RetrievalBoster BioCatalog #AR0024
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Sample Preparation - Paraformaldehyde Cooling and Dehydration
Sample Preparation - Paraformaldehyde Cooling and Dehydration
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
Wash the tissue thoroughly with PBS to remove blood. Use forceps to remove connective tissues.
Cut the tissue into slices with a thickness of 3 mm or less.
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Immerse the slices in 4% paraformaldehyde (pre-cool at 4°C) for 6 to 7 hours. The paraformaldehyde volume should be 20X greater than the tissue volume by weight.
Wash the tissue 3X with PBS (1 minute each).
Dehydrate the tissue by immersing the tissue sequentially as follows:
- 1X into 80% ethanol (1 hour at 4°C)
- 1X into 90% ethanol (1 hour at 4°C)
- 3X into 95% ethanol (1 hour each at 4°C)
- 3X into 100% ethanol (1 hour each at 4°C)
- 3X into dimethylbenzene (0.5 hr each at room temperature)
Sample Preparation - Liquid Paraffin Section
Sample Preparation - Liquid Paraffin Section
Prepare the first portion of liquid paraffin in a suitable bath and allow the paraffin to reach and maintain at 60°C.
60 °C
Immerse the tissue 2X into the paraffin bath (2 hours each).
Prepare the second portion of liquid paraffin in a suitable bath and allow the paraffin to reach and maintain at 60°C.
60 °C
Pour the second portion of paraffin into a mold.
Quickly transport the tissue from the paraffin bath to the mold with paraffin.
Incubate the tissue at room temperature until it coagulates.
Store the tissue at 4°C.
4 °C
Sample Preparation - Section Slicing and Incubation
Sample Preparation - Section Slicing and Incubation
Secure the paraffin section on the slicer.
Slice one to two pieces of the section to adjust the slicer so that the section and blade are parallel
Slice the remaining section carefully with ~5 µm thickness.
Incubate the sliced section in 40 to 50°C water to unfold.
Mount the tissue section onto Poly-Lysine (AR0003, Boster Bio) or APTES (AR0001, Boster Bio) coated glass slides.
Poly-L-Lysine Solution, 10X, For 100-200 SlidesBoster BioCatalog #AR0003
3-Aminopropyltriethoxysilane (APTES) 10ml 50X ConcentratedBoster BioCatalog #AR0001
Incubate the slides overnight at 37°C.
37 °C
Note
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.
Dewaxing/Deparaffinization
Dewaxing/Deparaffinization
Prepare the following reagents:
- 90% dimethylbenzene
- 95% dimethylbenzene
- 100% dimethylbenzene
- 90% ethanol
- 95% ethanol
- 100% ethanol
Sequentially immerse paraffin sections into:
- 90% dimethylbenzene (for 7 minutes)
- 95% dimethylbenzene (for 7 minutes)
- 100% dimethylbenzene (for 7 minutes)
- 90% ethanol (for 7 minutes)
- 95% ethanol (for 7 minutes)
- 100% ethanol (for 7 minutes)
Wash the slides with water to remove the ethanol.
Note
The process of dewaxing should be done in a fume hood at room temperature in summer. When the temperature is lower than 18°C, it is recommended to dewax at 50°C.
Inactivation
Inactivation
Immerse dewaxed paraffin section into the 3% H2O2 (AR1108, Boster Bio) at room temperature for 10 minutes.
3% Hydrogen Peroxide (H2O2) Solution, Lab GradeBoster BioCatalog #AR1108
Wash the section 3X to 5X with distilled water (total 3 to 5 minutes).
Antigen Retrieval (Heat Induced Epitope Retrieval: HIER)
Antigen Retrieval (Heat Induced Epitope Retrieval: HIER)
Citrate Buffer Powder 2L/Pack, For Heat Antigen RetrievalBoster BioCatalog #AR0024
Heat the buffer in the microwave and turn it off when the buffer has boiled.
Keep the boiled buffer in the microwave for 5 to 10 minutes.
Repeat the heating as outlined above for 1 to 2 times.
Cool the slides until it reaches room temperature.
Wash the sections 1X to 2X with PBS.
Blocking
Blocking
Add 5% BSA blocking solution or normal goat serum to the HIER treated samples.
Incubate the samples at 37°C for 30 minutes.
37 °C
Discard extra liquid (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 to the samples and incubate overnight at 4℃ or at 37℃ for 1 hour.
Wash the samples 2X with PBS (20 minutes each).
Secondary Antibody Incubation
Secondary Antibody Incubation
Dilute biotinylated secondary 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 to the samples and incubate at 37°C for 30 minutes.
37 °C
Wash the samples 2X with PBS (20 minutes each).
Staining
Staining
Incubate the samples at 37°C for 30 minutes.
37 °C
Wash the samples 3X with PBS (20 minutes each).
Add a suitable amount of DAB reagent (AR1022, AR1025, Boster Bio) to the samples and incubate in darkness at room temperature for 10 to 30 minutes.
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
Monitor the tissue staining intensity under a bright-field microscope.
Note
If the staining background is too high, wash the section 4X with 0.01-0.02% TWEEN 20 PBS and 2X with pure PBS after the SABC reaction and before DAB staining. Then use DAB to stain the samples.
Wash the samples 3 to 5 times with distilled water.
Counterstain (if necessary)
- Dehydrate
- Immerse the paraffin sections 2X in dimethylbenzene (7 minutes each)
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Check the tissue staining intensity under a bright-field microscope.