Immunohistochemistry (IHC) protocols allow localization and measurement of specific proteins in tissue. Paraffin-embedded tissue sections sometimes allow for better preservation of tissue morphology. Paraffin sections also allow you to work with larger tissues and store them more easily. Here we will show you how to perform IHC using paraffin-embedded sections (IHC-P). It consists of a series of procedures including fixation, paraffin embedding, sectioning, and staining of the tissue.
We provide you with a simple protocol for IHC-P that you can refer to, which contains some troubleshooting tips and hints. These methods for paraffin section preparation and immunohistochemical staining should help put you on the right experimental track.
|Stages||Solutions and Reagents|
|Section Preparation||Washing buffer, neutral buffered formalin, ethanol solution, xylene solution, liquid paraffin, antigen retrieval buffer|
|Antibody Staining||Blocking buffer, primary antibody, labeled secondary antibody, dilution buffer, washing buffer, incubation buffer, chromogen|
|Mounting||Mounting solution, washing buffer|
Prepare freshly dissected tissues and wash and cut them for use. Soak the tissue with a fixative for fixation. Neutral buffered formalin (NBF) is the usual fixative and the fixation time depends on the size of the tissue block and the type of tissue. Wash the tissues with buffer at the end of fixation. Dehydrate the tissues by soaking them several times with gradient concentrations of ethanol. Then wash the tissue several times using xylene. And immerse the tissue several times in liquid paraffin and keep it warm. Pour the paraffin wax into a mold, then transfer the tissue quickly into the mold and cool it at room temperature until the paraffin wax solidifies. Slice the paraffin-embedded tissues and incubate the sections in water to spread them. Mount the tissue sections on slides, dry them, and set them aside.
Incubate the slides in xylene solution and repeat several times to ensure that the sections are completely dewaxed. Transfer the slides to ethanol solution and wash them several times at reduced concentrations. Then wash with washing buffer to remove the ethanol. Be sure not to allow the slide to dry out during this process.
Perform antigen retrieval to reveal antigenic epitopes. The most commonly used antigen retrieval is heat-induced epitope restoration (HIER). There are various methods of heating after the addition of antigen retrieval buffer, such as microwaving, boiling, etc. You can read more about antigen retrieval to help determine the best conditions for your sample. When finished, cool the slide and wash it.
Select the ideal blocking reagent and incubate the sample with the blocking buffer. Incubation time and temperature are set on a case-by-case basis. When finished, wash the slide and remove the blocking buffer. For direct labeling, dilute the primary antibody appropriately and transfer the slide to the incubation buffer containing the primary antibody for incubation. Then wash the slides. For indirect assays, transfer the sections to the incubation buffer containing the secondary antibody and continue incubation. Wash the slide after completion to remove excess solution. Add the appropriate dilution of chromogenic agent to the slides and incubate away from light.
The counterstaining step is optional. Counterstaining provides contrast with antibody staining to better distinguish the target signal. Select the ideal counterstains, incubate the sections, and rinse them upon completion.
After all staining, use the appropriate sealing solution to mount coverslips and seal the samples. Mounted slides can be stored permanently at room temperature. Observe the color of the antibody stain in the tissue section under the microscope.
IHC-P is a procedure for sectioning and staining tissue embedded in paraffin. Although it sounds easy, the reality is that there are many possible combinations of tissue types, antibodies, and assays, which can easily lead to various problems. If you need to perform some troubleshooting, please refer to our extensive troubleshooting guide. We solve your problem by adjusting a relatively small number of variables.
Tissue section problem
No or inadequate staining
We hope that this information about IHC-P above is useful to you and useful as a reference guide. If you need more help with your experiments, please contact us for further assistance.
For research use only. Not intended for any clinical use.