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.

Quote Request

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.

Frequently Asked Questions (FAQs)

• Which cell type do you use?

  Human embryonic kidney 293 (HEK293) suspension cells.

• Can you use another cell type besides HEK293 cells?

  It may be possible. When filling out the quote request form (via the button provided above), you can add your request to the "Comments" section. Please indicate the cell line that you would prefer and whether you can provide that cell line to us. Should different cells be used, the cost of this service will be higher compared to using HEK293 cells.

• How many cells are in a pellet?

  Approximately 90 million cells. More cells available upon request.

• Can you provide a negative control?

  Yes, we can provide a pellet of fixed cells that have not been transfected with the gene-of-interest.

• How many sections can I obtain with a cell pellet?

  The number of sections per pellet will depend on the width of the section and how many cells are included. However, if the width of the section is ∼5 um, approximately 200 sections can be obtained with 1 standard cell pellet containing 90 million cells.

• What if I don't want my cells to be fixed in formaldehyde ("formalin-fixed")?

  We can fix cells using other methods. Send the desired protocol for fixing cells and we will quote accordingly.

• Why can the cell pellet be handled at BSL1?

  First, the HEK293 cells that are employed in this service are fixed, which renders them dead and noninfectious. Second, the protein-of-interest is produced recombinantly. That is, even if the protein-of-interest represents a protein from a pathogen (e.g., SARS-CoV-2), it is missing the other pathogen components to be infectious. Appropriate personal protective equipment (PPE), such as gloves, protective clothing, and safety goggles, should still be employed while handling the cell pellet because toxic chemicals were used to fix the cells.

  Notably, HEK293 cells, from which these suspension cells were derived [Malm, et al. Scientific Reports (2020)], are negative for various viruses, including the hepatitis B virus (HBV), hepatitis C virus (HCV), Epsteinn Barr virus (EBV), human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV) I and II, and squirrel monkey retrovirus (SMRV)[Uphoff CC, et al. J Biomed Biotech (2010)].

• What is the lead time?

  This depends on which phases are required to complete this service. The lead time ranges from 2 weeks to 2 months. The longer lead times reflect services in which we need to perform all 4 phases. See also "Outline of IHC Controls Service."

• Do you guarantee that my protein-of-interest will be expressed?

  We take several steps to increase the chance that your protein-of-interest will be expressed successfully. First, we review the protein sequence to identify any domains that should be excluded from expression. For example, we highly recommend that gene sequences encoding for transmembrane domains are not included since these domains can cause downstream issues. Second, we perform codon optimization for the expression system. Third, we perform DNA sequencing to ensure that the protein that we express is the protein that you requested.

  The level of protein expression is protein-dependent and cannot be predicted, which is why Phase 3 is included in this service (see "Outline of IHC Controls Service"). Therefore, we cannot guarantee that your protein-of-interest will be expressed successfully. Should your protein fail to be expressed, the service will not continue to Phase 4. You will only be billed for the work that was completed (Phases 1 - 3).

• Are all four phases of this service necessary? (see "Outline of IHC Controls Service")

  Not necessarily. Sometimes we already have the cells transfected with the gene-of-interest, such that only Phase 4 is required. In other cases, customers can provide the gene or cloned expression vector to us.

  Once Phases 1 - 3 are performed, they never have to be performed again.

• 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")

  No. Once cells have been produced that express Protein X, only Phase 4 needs to be performed and paid for.

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