I‘ve been using an unconjugated mouse anit rat anitbody to stain stem cells. The antibodies are scarce so unconjugated format is the onyl one we could obtain. we used a secondary antibody FITC. For controls I used an isotype fitc mouse Ig1.
I determined the positive cell populations from the isotype control.
However, was it necessary to add a secondary antibody(FITC) only control to determine the positive cell populations?
Hello Adamska Mr
To optimize your staining, both the primary and secondary antibodies should be titrated to ensure minimum background with a maximal specific signal. Fluorescently labeled secondary antibody only control should be used when indirect staining is performed. This control is important for determining whether there is any non-specific binding from the secondary antibody.
To make sense of your data and to draw accurate conclusions, it is important to include proper controls. The below attached link will provide you with more information on flow cytometry controls.
https://www.fortislife.com/products/antibody-resources/flow-cytometry-controls/flow-cytometry-controls
Best.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes