In indirect staining, the primary antibody is not fluorochrome-labeled, but is detected by a fluorochrome-labeled secondary antibody. This second reagent may be an antibody with specificity for the first antibody. Alternatively, the avidin-biotin system can be used, whereby an antibody is conjugated to biotin and detected with fluorochrome-labeled avidin.
With the wide range of conjugated antibodies now available, this method means that unconjugated primary antibodies raised against many different targets can be used in conjunction with a labeled secondary antibody for FACS analysis. This widens the choice of target proteins for the researcher.
When using secondary antibodies - All the primary antibodies have been raised in different species to ensure cross labeling via the secondary antibodies is not encountered. The secondary antibodies recognize one of the species exclusively.
More than one labeled antibody is bound per antigen molecule; The signal intensity could be amplified on a single epitope.
Different primary antibodies can be used with a single labeled secondary antibody; The reservoir of primary antibodies suitable for flow cytometry would be broadened for no limitation of fluorochrome labeling.
Fewer labeled antibodies are required.
|Human 14-3-3 beta / YWHAB Antibody||Human B7-DC / PD-L2 / CD273 Antibody|
|Human 14-3-3 sigma / Stratifin / YWHAS Antibody||Human B7-H3 / CD276 Antibody|
|Mouse 4-1BBL / CD137L / TNFSF9 Antibody||Human BACE1 Antibody|
|Human 4E-BP1 / EIF4EBP1 Antibody||Human Bcl-XL / BCL2L1 Antibody|
|AcMNPV Envelope glycoprotein gp64 / AcmNPV-gp64 Antibody||Human BID Antibody|
|Human ACY1 / Aminoacylase-1 Antibody||Human BLyS / TNFSF13B / BAFF Antibody|
|Human ADAM15 / MDC15 Antibody||Human BMP-4 Antibody|
|Human AK4 / Adenylate Kinase 4 / AK3L1 Antibody||Human c-MET / HGFR Antibody|
|Human AKR1B1 Antibody||Human C1QBP / HABP1 Antibody|
|Human ALDH1 / ALDH1A1 / ALDC Antibody||Human C4.4A / LYPD3 Antibody|
|Human ALDH7A1 Antibody||Human CALML3 Antibody|
|Human Angiotensinogen / SerpinA8 / AGT Antibody||Human Carbonic Anhydrase XII / CA12 Antibody|
|Influenza Anti-H7N9 Hemagglutinin / HA Antibody||Human Caspase-14 / CASP14 Antibody|
|Human Apolipoprotein H / APOH Antibody||Human CD111 / Nectin-1 / PVRL1 Antibody|
|Human ARD1 Antibody||Human CD116 / GM-CSFR Antibody|
|Human ASAM / CLMP Antibody||Human CD131 / CSF2RB / IL3RB / IL5RB Antibody|
|Human ASF1B Antibody||Mouse CD132 / IL2RG Antibody|
|Human ASGR2 / ASGPR2 Antibody||Human CD14 Antibody|
|Human B2M / beta-2 microglobulin Antibody|