In the Systemic Synuclein Sampling Study (S4)—a cross-sectional observational study of 59 participants with early, moderate, or advanced Parkinson disease (PD) and 21 healthy controls (HCs)—Dr. Chahine et al. found lower total α-synuclein levels in the CSF of patients with PD compared with HCs with a reasonable sensitivity of 87%, but this finding had low specificity. On the other hand, α-synuclein immunoreactivity in skin and submandibular gland was specific for PD but not sensitive. In response, Dr. Gibbons et al. cite previous studies that reported much higher sensitivities (80%–95% vs 24.1%) for the detection of α-synuclein in the skin in patients with PD. They argue that this discrepancy cannot be explained by inclusion of late-stage PD in such studies, citing high-detection rates of phosphorylated α-synuclein in patients with early-stage PD and REM sleep behavioral disorder (RBD), and low-false positivity. They propose that the discrepant results in the S4 study may be explained by the study’s methodology of formalin fixation of the skin biopsies, which they claim has not gained acceptance in the study of peripheral nerve tissue because of the diminished integrity of peripheral antigen retrieval; paraffin embedding of the tissue, which they argue provides only a fraction of the volume obtained with larger frozen tissue sections; and automated immunohistochemical staining. They suggest that future studies in this area should use more accepted standardized methods for processing skin biopsy tissue for phosphorylated α-synuclein. Responding to these comments, the authors suggest that previous conflicting results have primarily been due to relatively low levels of study rigor in assessing the accuracy of the various immunohistochemistry methods, which, in the S4 study group, included multiple independent slide-reading judges, third-party blinding of such judges, and validation against gold standard neuropathologic diagnosis. They agree that reports of high sensitivity of peripheral α-synuclein detection in patients with idiopathic RBD are encouraging for the early detection of α-synucleinopathies but argue that not all patients with PD have preceding RBD and that those who do tend to have more widespread and severe brain synucleinopathy. They counter that technical differences in paraformaldehyde and formalin fixation are minimal and cite previous methods from S4 authors supporting the use of formalin-fixed, paraffin-embedded (FFPE) tissue. They also argue that the multiple S4 tissue sections that they assessed for each tissue site and subject resulted in sufficient tissue volumes to overcome any limitations of individual paraffin-embedded samples. They note that thick sections and immunofluorescent signal development methods require rare technical expertise, whereas FFPE methods and autostainers are more widely available, with autostaining methods also providing greater replicability and potentially better long-term storage than free-floating immunohistochemical methods. This exchange highlights enduring methodological uncertainties, tradeoffs, and debates regarding the detection of antigens such as synuclein in tissue samples, which need to be more definitively resolved before such detection is adopted into clinical practice.