Immunohistochemistry (IHC) Controls

Fixed positive control cells for IHC

RayBiotech provides fixed cells containing the overexpressed protein-of-interest, which can be used as positive controls in immunohistochemistry (IHC) experiments or to validate antibodies for IHC use. The cells are provided as a pellet, which can then be cut into sections for IHC analysis (see Figure 1, "General IHC Steps").

IHC is a valuable tool in research, clinical diagnosis, and drug development since it enables the visualization of one or more proteins-of-interest in cells or tissues. However, obtaining IHC positive controls can be difficult, particularly when the protein-of-interest is produced by a pathogen.

IHC controls analysis
Figure 1. IHC analysis of fixed HEK293 cells stained with a mouse monoclonal IgG antibody to the SARS-CoV-2 Spike S2 subunit. A) Untransfected cells. Cells transfected with SARS-CoV-2 Nucleocapsid (B), full-length Spike S1 Subunit (C), and full-length Spike S2 Subunit (D). Fixed cells were obtained by RayBiotech.

Service Features

  • ✔ Customizable: choose the target protein you want
  • ✔ Formalin-fixed human cells
  • ✔ Cells can be handled at biosafety level 1 (BSL1)
  • ✔ Negative controls available
  • ✔ Affordable: ∼200 sections can be obtained per cell pellet

Outline of IHC Controls Service

Phase 1: Gene Synthesis

If your gene-of-interest (GOI) is not available, we will synthesize the GOI with codon optimization that is appropriate for the expression system. Larger genes will result in longer lead times.
Lead time: 2 – 4 weeks
Deliverables: Gene will remain at RayBiotech for Phase 2. If desired, the gene can also be shipped to the customer.

Phase 2: Gene Cloning

GOI is cloned into an expression vector specific for the expression system. Proper insertion of the gene is confirmed via DNA sequencing.
Lead time: 1 – 2 weeks
Deliverables: Cloned gene will remain at RayBiotech for Phase 3.

Phase 3: Small Scale Gene Expression

Expression of GOI is verified with a western blot using a small number of cells.
Lead time: 1 week
Deliverables: Customer will receive the western blot image. Gene sequencing data available upon request.

Phase 4: Large Scale Gene Expression & Cell Fixation

GOI is expressed in ∼90 million cells. Cells are pelleted and fixed.
Lead time: 2 weeks
Deliverables: Pellet of fixed cells will be shipped to customer with ice.

General IHC Steps

  1. Fixation: Cells or tissues are "fixed" using chemicals that kill the cells and lock their activity in place. It also enables large molecules like antibodies to access intracellular proteins easily.
  2. Slicing: Cell pellets or tissue samples are cut into very thin slices that are microns thick.
  3. Adherence: The cells or tissue are adhered to a glass slide and dried.
  4. Primary Antibody: A primary antibody that binds to the protein-of-interest is added.
  5. Secondary Antibody: A secondary antibody conjugated to a detection reagent, such as horseradish peroxidase, is then added, which binds to the primary antibody.
  6. Detection: The location of the protein-of-interest is visualized via the secondary "detection" antibody.
  7. Analysis: The presence, amount, and location of the protein-of-interest are examined by a pathologist.
Lab worker with a slide in their hands

Frequently Asked Questions (FAQs)

  • Which cell type do you use?
  • Can you use another cell type besides HEK293 cells?
  • How many cells are in a pellet?
  • Can you provide a negative control?
  • How many sections can I obtain with a cell pellet?
  • What if I don't want my cells to be fixed in formaldehyde ("formalin-fixed")?
  • Why can the cell pellet be handled at BSL1?
  • What is the lead time?
  • Do you guarantee that my protein-of-interest will be expressed?
  • Are all four phases of this service necessary? (see "Outline of IHC Controls Service")
  • I previously had you make fixed cells for me with Protein X. I need another cell pellet that expresses Protein X. Do I need to pay for all four phases of this service again? (see "Outline of IHC Controls Service")

References

  • Szabolcs M, et al. Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues. Appl Immunohistochem Mol Morphol. 2021;29(1):5-12. doi:10.1097/PAI.0000000000000878