IHC Controls

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.

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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

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

Discuss your project requirements

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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

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.
Slicing: Cell pellets or tissue samples are cut into very thin slices that are microns thick.
Adherence: The cells or tissue are adhered to a glass slide and dried.
Primary Antibody: A primary antibody that binds to the protein-of-interest is added.
Secondary Antibody: A secondary antibody conjugated to a detection reagent, such as horseradish peroxidase, is then added, which binds to the primary antibody.
Detection: The location of the protein-of-interest is visualized via the secondary "detection" antibody.
Analysis: The presence, amount, and location of the protein-of-interest are examined by a pathologist.

Frequently Asked Questions

Still have questions?

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?

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?

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")

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")