Why is it important to include both positive and negative controls in your IF experiment?

Positive and negative controls are at the heart of any good experiment. Without them, it is difficult to assess root causes when troubleshooting. Positive controls should be included to demonstrate:

• Efficacy and potency of treatment-induced modulation
• Secondary antibody quality
• That processing steps were executed effectively
• That buffers are in good working order

Effective negative controls may include:

• Secondary antibody alone to assess its contribution to overall signal
• Isotype control to determine if anything in the sample causes non-specific primary antibody binding 
• Cells/tissue only to set a baseline for sample autofluorescence
• A cell line or tissue with known negative-expression for the target of interest

Every IF staining experiment we perform at CST includes the following elements:

1. S6 Ribosomal Protein (5G10) Rabbit mAb #2217 as a robust readout for both quality of fixation/processing and secondary antibody health.
2. Secondary antibody only and cells/tissues alone controls
3. A treatment-appropriate control, when applicable.

Last updated: September 12, 2024