Immunohistochemistry (IHC) Protocol
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.
- 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.
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.
- Dissect the tissue, 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 before Fixation Procedure:
- 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.
Protocol for Fixed and Paraffin Embedded Tissues with Sodium Citrate Antigen Retrieval:
Formalin or other aldehyde fixation forms 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.
- Fix tissue with formalin, or other fixative, then embed in paraffin wax to dehydrate
- Cut tissue in 4 µm sections on charged slides and heat in oven 45 min at 60°C
- Wash slides 3X in xylene for 5 min at RT
- Wash slides 3X in 100% alcohol for 3 min at RT
- Wash slides 2X in 95% alcohol for 3 min at RT
- Wash slides 1X in 80% alcohol for 3 min at RT
- Gently rinse slides using distilled H2O for 5 min at RT
- Quench endogenous peroxidase for 10 min with 3% H2O2 in 0.1 M PBS and wash 3 times with distilled water, 2 minutes each
Sodium Citrate Antigen Retrieval (for other antigen retrieval methods, please see Aviva's IHC Tips and Tricks)
- Retrieve epitopes by incubating the sections at 98-100° C in a microwave in 0.01 M citrate buffer (pH 6.0) for 15-20 minutes. Cool down in room temperature for 30-60 min
- Rinse 1X in 0.1% TBST for 1 min at RT
(Tissues should not dry at any time during the procedure!
- Apply universal protein block for 20 min at RT; drain
- Apply (diluted) primary antibody for 45 min at RT (this may require optimization); rinse 3X for 1 min in 0.1% TBST
- Apply (diluted) biotinylated secondary antibody for 45 min at RT; rinse 3X for 1 min in 0.1% TBST
- Apply AP-streptavidin for 30 min at RT; rinse 3X for 1 min in 0.1% TBST
- Apply alkaline chromogen substrate for 30 min at RT (may require optimization); rinse 1X in dH2O
- Add counterstain for 10-30 seconds with a hematoxylin counterstaining solution
- Rinse 1X in dH2O
(Chromogen substrate has to be alcohol insoluble for this method to work!
- Wash slides 2X in 80% alcohol for 1 min at RT
- Wash slides 2X in 95% alcohol for 1 min at RT
- Wash slides 3X in xylene for 1 min at RT
- Apply coverslip with mounting medium