Feb 07, 2018
Version 2
IHC-F Protocol V.2
- CJ Xia1
- 1University of Virginia, Charlottesville
- Boster Bio
Protocol Citation: CJ Xia 2018. IHC-F Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.m4hc8t6
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 06, 2018
Last Modified: March 28, 2018
Protocol Integer ID: 10089
Keywords: ihc, ihc-f, immunohistochemistry, frozen, immunostaining, immunohistochemistry method with frozen tissue section, immunohistochemistry method, immunological technique, powerful assay for protein detection, localization of specific cellular component, protein detection, specific cellular component, assay, frozen tissue section, powerful assay, cell
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 frozen 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
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
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
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Protocol materials
Antibody Dilution Buffer With BSA, Reducing BackgroundBoster BioCatalog #AR1016
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
Hematoxylin Counterstain SolutionBoster BioCatalog #AR0005
Phosphate Buffered Saline (PBS) PowderBoster BioCatalog #AR0030
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
Troubleshooting
Sample Preparation
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.
Sample Preparation - Snap Freezing and OCT Embedding
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 the thickness of 3 mm or less.
Immediately snap freeze the tissue in iso-pentane cooled in dry ice and keep the tissue at -70°C. Do not allow frozen tissue to thaw before cutting.
-70 °C
Prior to cryostat sectioning, position the tissue in a mold (which can be simply made by using tin foil) and cover the tissue completely in Optimal Cutting Temperature (OCT) embedding medium.
Use forceps to take the bottom part of the mold into liquid nitrogen for 1 to 2 minutes. The OCT should change to white.
Sample Preparation - Cryostat Sectioning
Pre-cool a slicer box and detector to -22°C and -24°C respectively. Ensure the completeness and smoothness of the blade.
Place the tissue from the mold to the detector where the tissue is fixed.
Quickly and carefully slice the cryostat sections at 5-10 µm and mount them on gelatin-coated histological slides. Note that:
- Use coverslip to take sliced tissue
- Cryostat temperature should be between -15°C and -23°C
- The sections will curl up if the specimen is too cold
- The sections will stick to the knife if the specimen is too warm
Air dry the sections at room temperature for 30 minutes to prevent them from falling off the slides during antibody incubations.
Store the slides at -70°C. Note that:
- The slides can be stored unfixed for several months at -70°C
- Frozen tissue samples saved for later analysis should be stored intact
-70 °C
Immediately add 50 µL of ice-cold fixation buffer to each tissue section upon removal from the freezer.
Fix frozen section by immersing it into 4% paraformaldehyde (AR1068, Boster Bio) at 2-8°C for 8 minutes (or optimally at -20°C for 20 minutes).
4% Paraformaldehyde (PFA) Solution in PBSBoster BioCatalog #AR1068
Wash the section 3X with PBS and allow it to dry at room temperature for 30 minutes.
Inactivation
Mix H2O2 with distilled water (v/v: 1:50).
Immerse frozen sections or cell climbing slices into the diluted H2O2 at room temperature for 10 minutes
Wash the section 3X with distilled water (1 minute each).
Antigen Retrieval (Proteolytic Induced Epitope Retrieval: PIER)
Dry the frozen sections with filter paper.
Add compound digestion solution (AR0022, Boster Bio) (e.g. Trypsin solution or other enzymatic antigen retrieval solution) to the sections or slices.
Antigen Retrieval Buffer (Enzymatic Digestion) For IHCBoster BioCatalog #AR0022
Incubate the sections at room temperature for 3 to 5 minutes.
Wash the sections with 3X PBS (5 minutes each).
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
Discard extra liquid (No washing required).
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 at 37°C for 30 minutes.
37 °C
Wash the samples 2X with PBS (20 minutes each).
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
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 (AR1025, AR1022, Boster Bio) to the samples and incubate in darkness at room temperature for 10 to 30 minutes.
DAB Chromogenic Substrate Reagent Kit (Blue)Boster BioCatalog #AR1025
DAB Chromogenic Substrate Reagent Kit (Yellow)Boster BioCatalog #AR1022
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 3X to 5X 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.