IHC-frozen is essential in research and diagnostics due to its superior preservation of protein antigenicity and cellular morphology, ensuring accurate detection of specific antigens and detailed observation of tissue structures. It allows for the analysis of fresh tissue samples, making it valuable for studying dynamic biological processes and sensitive samples, offering insights closer to their natural state. The technique's rapidity is crucial in situations requiring quick results, such as intraoperative consultations, aiding surgeons and pathologists in making timely diagnostic and treatment decisions. IHC-frozen's compatibility with specific antibodies enhances its accuracy in protein localization and detection. Its versatility extends to various research applications, including gene expression studies, signaling pathway investigations, and enzyme histochemistry, providing researchers with a comprehensive tool for exploring multiple parameters within a single tissue sample.
Benefits Include:
Preservation of Antigenicity: IHC-frozen maintains the antigenicity of proteins and biomolecules better than formalin-fixed, paraffin-embedded sections, ensuring accurate and reliable detection of specific antigens with antibodies.
Superior Cellular Morphology: Frozen sections offer excellent preservation of cellular morphology, allowing for detailed observation of cellular structures and tissue architecture, which is critical for research and diagnostic purposes.
Analysis of Fresh Tissue: IHC-frozen permits the analysis of fresh tissue samples, making it ideal for studying dynamic biological processes and samples sensitive to fixation, providing insights closer to their natural state.
Rapid Results: The technique allows for quick preparation and analysis of tissue sections, making it valuable for situations requiring swift results, such as intraoperative consultations during surgery and urgent diagnostic evaluations.
View our antibodies validated in house for use in IHC-Fr HERE
Protocol:
A. Solutions and Reagents
- Prepare 1X Tris Buffered Saline with Tween 20 (TBST).
- Use fresh 4% Paraformaldehyde (PFA).
- Prepare Blocking Buffer: Combine 10% Normal Goat Serum, 0.3M Glycine, and 0.25% TritonX-100 in TBST.
- Utilize a Fluorochrome-conjugated Secondary Antibody compatible with the primary antibody's host species (e.g., rabbit).
- Have Antifade Mounting Medium ready.
B. Tissue Preparation
- After tissue dissection, immerse it in 4% PFA with 15% sucrose for 24 hours at 4°C. Subsequently, dehydrate the tissue and immerse it in 30% sucrose solution at 4°C until it settles at the bottom.
- Completely encase the tissue in OCT compound.
- Slice the tissue into 10-20 µm sections and mount them on slides. Store the slides at -20°C until the staining procedure.
C. Immunostaining
- Allow the tissue sections to air-dry for 30 minutes at room temperature.
- Optionally, fix the sections in 4% PFA for 10 minutes at room temperature.
- Wash the sections in 1X TBST three times for 3 minutes each.
- Block the sections with Blocking Buffer for 30 minutes at room temperature.
- Drain the slides without rinsing and apply the primary antibody, diluted in 1X TBST. Incubate overnight at 4°C.
- Rinse the slides three times in 1X TBST for 3 minutes each.
- Incubate the specimens with the fluorochrome-conjugated secondary antibody (with DAPI 1/10,000 or Hoechst 33258), diluted at 1/1000 in 1X TBST for 2-3 hours, protected from light.
- Rinse the slides three times in 1X TBST for 3 minutes each.
- Mount the specimens with an antifade mounting medium.
- Visualize the samples using a fluorescence microscope.