Did you already waste time trying to find the right antibody for your immunofluorescence experiments? Get a guaranteed one thanks to our antibody generation service for immunofluorescence. Our platform of hybridoma generation for diagnostic applications has an unrivaled success rate thanks to our strong expertise in various immunization approaches, making us the best partner for your research!

How could you be sure that your antibodies will work in your immunofluorescence experiment?

Don’t waste time in testing several antibodies in your application! To save your time and your money, ProteoGenix launched a hybridoma development service guaranteed for immunofluorescence applications. Want to be sure to get the prefect antibody? Follow these 3 steps:

  • Let us manage the whole antibody development process from antigen design to final production
  • We test the antibodies in your OWN conditions
  • We send you part of the sample so that you can validate it by yourself in your own laboratory. You’re fully charged only if you’re satisfied!

Ready to start? Contact our PhD account managers who will guide you during your project!

Step Content Timeline Deliverables
Antigen production
  • Antigen design
  • Gene synthesis
  • Test of two expression systems
  • Antigen production
4 to 11 weeks
Mice immunization
  • Mice injections
  • Immune response control by IF
7 to 11 weeks
Hybridoma production
  • Mice selection
  • Screening of the fusion products by IF
  • Test of two expression systems
  • Specificity control
2 to 4 weeks
  • 10-20 best clones for customer’s selection
Antibody production
  • Subcloning of two parental clones
  • Antibody production and purification
7 to 11 weeks
  • 100µg of purified antibody + 50µg of purified protein to confirm hybridoma purchase

Deliverables after total payment:

  • 1.9mg of purified antibody
  • One hybridoma cell line (4 frozen vials)
  • 150µg of purified protein
  • Gene in pUC57

Labeled primary or secondary antibody for immunofluorescence?

Direct staining refers to the use of a labeled primary antibody. Direct staining facilitates multiplexing as it allows using primary antibodies generated in the same species.

Indirect staining corresponds to the use of labeled secondary antibodies and is based on the pairing of a primary with a secondary antibody. Usually, the secondary antibody is specific for the host species of the primary antibody limiting the multiplexing application. Indirect staining is generally preferred for low abundance target antigen as it demonstrates higher sensitivity compared to direct staining as several secondary antibodies can bind a single primary antibody.

Any doubt about how to make the best choice for your project? Our account managers are available to answer to your questions.

How should you control your antibodies for immunofluorescence?

As every scientific experiment, immunofluorescence requires controls to make sure that your antibody is correctly detecting its target and not to observe false-positive results.

Here are some of the controls that are regularly undertaken in immunofluorescence.

Positive controls

Positive controls can be obtained by testing a cell line known to express your protein of interest.

Negative controls

Commonly used negative controls include:

  • Tests on cell lines known not to express the protein of interest such as KO cell lines or cells with gene mediated silencing.
  • No primary control: in the “no primary control”, the assays conditions remain exactly the same as the “normal” experimental conditions except that the primary antibody is not added. The goal of this control is to test the specificity of the primary antibody.
  • Isotype control: the isotype control is commonly used to determine the background level of the sample. The isotype control necessitates an antibody of the same isotype, clonality, label and host species of the primary antibody but should detect a molecule not present in the sample. The experimental conditions should be identical to those used with the primary antibody.

Immunofluorescence principle and the role of antibodies

Immunofluorescence is based on the use of an antibody-label conjugate to determine the localization and expression of target proteins in fixed cells or tissues. The antibody-label conjugates combine two properties which make them particularly suitable for microscopy experiments:

  • The specificity of the antibody allowing to detect only the targeted antigen
  • The “detectability” of the label

There are mainly two types of immunofluorescence:

  • Direct immunofluorescence which is based on the use of a primary antibody conjugated to a fluorophore
  • Indirect immunofluorescence which is based on the use of a labeled secondary antibody recognizing the primary.