After fixation and permeabilization, which flow cytometry antibodies can still be used to stain intracellular markers? Please recommend compatible fluorochromes.

A decline in PE signal is not always an antibody issue; excessive fixation time or overly strong permeabilization buffer can also damage the fluorochrome. It is recommended to fix at room temperature for 20-30 minutes or at 4°C overnight, followed by thorough washing, and not to exceed the time stated in the instructions by too much. Intracellular IL-17A staining should be performed after permeabilization, and the antibody diluent should be permeabilization buffer. If PE truly does not work, IL-17A can be switched to APC, and other surface markers can be assigned to FITC/PE. In multicolor panels, intracellular markers should preferably be assigned to stable fluorochromes such as APC/BV421, while surface markers will be brighter with PE.

After fixation and permeabilization, PE signal decreases, which may be related to the fixative, methanol permeabilization, or disruption of the clone epitope; not all PE will perform poorly. Prioritize small-molecule dyes that are tolerant to fixation and permeabilization: APC, Alexa Fluor 647, eFluor 660, BV421. BV421 is highly bright, but requires the instrument to have a violet laser; if your machine does not have a violet laser, do not buy it. APC is usually more stable than PE after fixation and permeabilization and is well compatible with permeabilization buffer, making it suitable for intracellular IL-17A. During staining, intracellular antibodies are generally added after permeabilization, and extending the incubation time to 30-45 minutes can improve the signal.

Common IL-17A clone numbers include eBio17B7, TC11-18H10.1, and BL168, which are relatively easy to purchase in China. The APC or Alexa Fluor 647 version of eBio17B7 performs relatively stably after fixation and permeabilization, and is sufficient for personal Th17 subsetting; BioLegend's BL168 APC is also acceptable, with slightly weaker brightness but still capable of separating populations. For the BV421 version, it is best to first confirm that the instrument has a 405 nm laser. Fixation and permeabilization strengths differ among kits, so it is recommended to first perform a small-scale comparison using APC and PE versions of the same clone. For 3-day in-stock availability in China, confirm the lot with the distributor; distributors in first-tier cities often stock common clones.