Eosinophils are granulated white blood cells that play an important role in mediating host immunity against fungal, bacterial, viral, and helminthic infections, as well as triggering the inflammatory cascade in several allergic diseases (Klion et al., 2020). These white blood cells are frequently found to be elevated among asthmatics, particularly in their peripheral blood and bronchial lavage fluid; their numbers in these locations broadly correlate with asthma severity (Hogan et al., 2008). Eosinophilic granules are primarily comprised of four cationic proteins - the major basic protein (MBP), eosinophil peroxidase (EPX), eosinophil derived neurotoxin (EDN), and eosinophil cationic protein (ECP) (Bystrom et al., 2011; Davoine and Lacy, 2014; Hogan et al., 2008).

Platelet activating factor (PAF) is a phospholipid that acts as an activator and chemoattractant of eosinophils through various mechanisms (Davoine and Lacy, 2014). By acting on the PAF receptor and through an unidentified PAF receptor-independent mechanism (Dyer et al., 2010), this phospholipid mediator can trigger eosinophil activation and degranulation, consequently resulting in the release of several of the aforementioned cationic proteins and cytokines during inflammatory responses (Menzies-Gow and Robinson, 2002; Pałgan and Bartuzi, 2015; Muñoz-Cano et al., 2019). Among the cytokines released from eosinophils are T helper 2 (Th2) cytokines including IL-4, IL-5, IL-9, and IL-13 (Davoine and Lacy, 2014). IL-9 and IL-13 in particular promote Th2 reactions during eosinophilic allergic responses (Gounni et al., 2000; Woerly et al., 2002; Phipps et al., 2002; Schmid-Grendelmeier et al., 2002; Spencer et al., 2009; Walsh et al., 2011; Akdis et al., 2020).

The intracellular distribution of eosinophil cytokines may be analyzed through immunofluorescence analysis. Immunofluorescence is a process through which cellular proteins can be detected and measured by conjugating fluorophores to antibodies. This technique has been used in numerous reports to detect eosinophil proteins and their localization during cell activation (Levi-Schaffer et al., 1995; Lacy et al., 1999; Kim et al., 2013). Unfortunately, the charged nature of eosinophil cationic proteins interferes with the detection of specific antibody binding to low abundance proteins, including cytokines, resulting in nonspecific antibody binding and producing images with highly fluorescent backgrounds (Mahmudi-Azer et al., 1998). This is compounded by endogenous autofluorescence in eosinophils which contain high levels of fluorescent flavins in their granules, particularly flavin adenine dinucleotide (Mayeno et al., 1992). Due to interference from nonspecific backgrounds, there is a need to improve the signal to noise by optimization of current protocols. Therefore, it is important to develop permeabilization and blocking techniques together with appropriate isotype controls that ensure the detection of test antibodies binding to their targets to indicate specific immunofluorescent labelling. This work aims to develop an optimized protocol for increased specificity of eosinophilic immunolabelling for enhanced intracellular protein detection. We also describe the procedures for automated intensity measurements and colocalization of intracellular markers using super-resolution microscopy.