Immunohistochemistry (IHC)
Description:
IHC is used to understand the distribution and localization of proteins in different parts of a biological tissue. IHC detects specific antigens in preserved tissue sections using an appropriate antibody labeling strategy. Samples are collected, fixed to maintain cell morphology, tissue architecture and antigenicity of target epitopes, and then sectioned. A variety of antibody staining schemes can produce informative IHC images. Having an antibody conjugated to a fluorophore (immunofluorescence) is a common detection method. See our validation data page for immunohistochemical data. Click for related immunocytochemistry protocol.
Formalin Immersion/Formaldehyde Perfusion Fixation of Tissues:
- Preserve tissue morphology and retain the antigenicity of the target proteins, fix the tissue by vascular perfusion using a formaldehyde fixative solution.
- Perfuse tissue with a sucrose solution and dissect the desired tissue.
Note: When it is not possible to fix by perfusion, dissected tissue may be fixed by immersion in a 10% formalin solution for 4 to 8 hours at room temperature. It is commonly accepted that the volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue antigens may either be masked or destroyed.
- Mount in OCT embedding compound, and freeze at -20 to -80 °C.
- Cut 5-15 µm thick tissue sections using a cryostat.
Note: The suggested cryostat temperature is between -15 and -23 °C. The section will curl if the specimen is too cold. If it is too warm, it will stick to the knife.
- Thaw-mount the sections onto gelatin-coated histological slides.
- Dry the slides for 30 minutes on a slide warmer at 37 °C. Slides containing cryostat sections can be stored at -20 to -70 °C for up to 12 months.
Cryopreservation of Tissues:
- Immediately snap freeze fresh tissue in isopentane mixed with dry ice, and keep at -70 °C. Do not allow frozen tissue to thaw before cutting.
- Embed the tissue completely in OCT compound prior to cryostat sectioning.
- Cut cryostat sections at 5-10 µm and mount on gelatin-coated histological slides.
Note: The suggested cryostat temperature is between -15 and -23 °C. The section will curl if the specimen is too cold. If it is too warm, it will stick to the knife.
- Air dry the sections for 30 minutes at room temperature to prevent sections from falling off the slides during antibody incubations.
Note: Slides can be stored unfixed for several months at -70 °C. Frozen tissue samples saved for later analysis should be stored intact.
- Immediately add 50 µL of ice-cold fixation buffer to each tissue section upon removal from the freezer.
- Fix for 8 minutes at 2-8 °C or, optimally, at -20 °C for 20 minutes.
General Immunohistochemistry Staining Procedure:
- When staining cryostat sections stored in a freezer, thaw the slides at room temperature for 10-20 minutes.
- Rehydrate the slides in wash buffer for 10 minutes. Drain the excess wash buffer.
Note: Excessive fixation may result in the masking of an epitope and strong non-specific background signal that can obscure specific labeling. If necessary, an antigen retrieval protocol can be performed at this time. However, many antigen retrieval techniques are too harsh for cryostat cut tissue sections.
- Surround the tissue with a hydrophobic barrier using a barrier pen.
- Block non-specific staining between the primary antibodies and the tissue by incubating in blocking buffer for 30 minutes at room temperature.
- Apply primary antibodies diluted in buffer according to manufacturer’s instructions. For fluorescent IHC staining of frozen tissue sections, it is recommended to incubate overnight at 2-8 °C. This incubation regime allows for optimal specific binding of antibodies to tissue targets and reduces non-specific background staining. These variables may need to be optimized for your system.
- Wash slides 3 times for fifteen minutes each in wash buffer.
- Incubate with the secondary antibody diluted in buffer according to the manufacturer’s instructions.
- Wash slides 3 times for fifteen minutes each in wash buffer.
- Add 300 µL of the diluted DAPI solution to each well, and incubate 2-5 minutes at room temperature. DAPI binds to DNA and is a convenient nuclear counterstain. It has an absorption maximum at 358 nm and fluoresces blue at an emission maximum of 461 nm.
Note: DAPI counterstain can obscure visualization of targets localized in cell nuclei.
- Rinse 1 time with 1X PBS.
- Mount with an anti-fade mounting media.
- Visualize using a fluorescence microscope.
Aviva System Biology's Protocol for Fixed and Paraffin Embedded Tissues with Sodium Citrate Antigen Retrieval:
Formalin or other aldehyde fixation form protein cross-links that mask the antigenic sites in tissue specimens, leading to weak or false negative staining for immunohistochemical detection of certain proteins. Sodium citrate treatments breaks the protein cross-links, unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, enhancing staining intensity of antibodies.
Tissue Fixation
- Tissue should be fixed with formalin followed by an embedding in paraffin wax
- Paraffin wax: used to dehydrate tissue
Tissue Sectioning
- Tissue should be cut into 4 µm sections on clean, charged microscope slides and then heated in a tissue-drying oven for 45 minutes at 60°C
Deparaffinization: At Room Temperature
- 5 minutes each: wash slides in 3 changes of xylene
Rehydration: At Room Temperature
- 3 minutes each: wash slides in 3 changes of 100% alcohol
- 3 minutes each: wash slides in 2 changes of 95% alcohol
- 3 minutes each: wash slides in 1 change of 80% alcohol
- 5 minutes: Gently rinse slides using distilled water
Antigen Retrieval:
- At 99-100°C for 20 minutes: steam slides in 0.01 M sodium citrate buffer, pH 6.0
- At Room Temp for 20 minutes: remove slides from heat and let cool in buffer
- At Room Temp for 1 minute: Rinse in 1x TBS with Tween (TBST)
Immunostaining: Tissues should not dry at any time during the procedure!
- At Room Temp for 20 minutes: apply universal protein block
- Drain the protein block from the slides
- 45 minutes at Room Temp: Apply (diluted) primary antibody
- 1 minute at Room Temp: Rinse slides in 1x TBST
- 30 minutes at Room Temp: Apply a biotinylated secondary antibody (specific to the host of the primary antibody)
- 1 minute at Room Temp: Rinse slides in 1x TBST
- 30 minutes at Room Temp: Apply alkaline phosphatase streptavidin
- 1 minute at Room Temp: Rinse slides in 1x TBST
- 30 minutes at Room Temp: Apply alkaline phosphatase chromogen substrate
- 1 minute at Room Temp: Wash slides with distilled water
Dehydration:At Room Temperature
- Chromogen substrate has to be alcohol insoluble for this method to work!
- 1 minute each: Wash slides in 2 changes of 80% alcohol
- 1 minute each: Wash slides in 2 changes of 95% alcohol
- 1 minute each: Wash slides in 2 changes of 100% alcohol
- 1 minute each: Wash slides in 3 changes of xylene
- Apply coverslip
For more information, see:
What is immunohistochemistry?
Other immunohistochemistry protocols at IHC world