The two main methods for performing immunohistochemistry include paraffin-embedded sections and frozen sections. Frozen sections are simple, and quick to prepare. Frozen sections are mostly used for fresh tissues, formaldehyde-fixed tissues, refrigerator frozen tissues, etc.
We describe general procedural guidelines for system preparation and immunohistochemistry-frozen (IHC-Fr), and the client can determine the application protocol based on the target antigen. In addition to the protocols used for IHC-Fr, we also list some useful tips and troubleshooting guides.
Stages | Solutions and Reagents |
Section Preparation | Freeze embedding medium, PBS, fixative, protease inhibitors, blocking buffer |
Antibody Staining | Blocking buffer, primary antibody, labeled secondary antibody, dilution buffer, PBS, incubation buffer, chromogen |
Mounting | Mounting solution, PBS |
Prepare freshly dissected tissue blocks on a tissue substrate and cover the entire block with the frozen embedding medium. Freeze the tissue block in liquid nitrogen, transfer the frozen tissue block to a frozen sectioning machine, and slice the frozen tissue block to the desired thickness. Place the tissue sections on slides and dry the tissue sections for use.
Fix the tissue section with a suitable fixative and immerse the slide in the pre-cooled fixative. Then pour off the fixative and rinse the slides. After fixation is complete, the slides are incubated in the blocking buffer to block the non-specific binding of tissue immunoglobulins. At the end of the incubation, the slide is rinsed to drain 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 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.
The following troubleshooting guide is intended to explain the causes and possible solutions to common problems observed in IHC-Fr staining. If you have any questions, please refer to our extensive troubleshooting guide.
Tissue section problem
No or inadequate staining
High background
We provide more information on IHC-Fr optimization protocols and troubleshooting to help you get the best results. Creative Biolabs also offers customized products and services to meet specific customer requirements, please contact us for scientific support.
At Creative Biolabs, we are dedicated to providing high-quality antibodies for various research applications. Each product in our extensive range has been rigorously tested to ensure superior reliability and efficacy. To showcase the performance of our antibodies, we have conducted numerous experiments using Immunohistochemistry-Frozen (IHC-Fr). Below, you will find a table listing a selection of our antibody products along with images from these experiments, demonstrating their proven reliability.
Product Name | Catalog Number | Target | Image | Description |
---|---|---|---|---|
Rabbit Anti-3-SLeX Recombinant Antibody (clone E2-B4) | VS9-YC2 | 3-SLeX |
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The antibody was uesd for staining of the FFPE oesophageal adenocarcinoma tissue. The dilution range of 10-40 μg/mL is recommended. |
Rabbit Anti-SSEA4 Recombinant Antibody (clone E2-F8) | VS9-YC1 | SSEA4 |
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The antibody was uesd for staining of the FFPE oesophageal adenocarcinoma tissue. The dilution range of 10-40 μg/mL is recommended. |
Reference
For research use only. Not intended for any clinical use.
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