Preservation of intracellular protein-protein interactions in archived cytological specimens: Optimization of preanalytical variables for in situ proximity ligation assay
Year: 2026
Type: preprint
Abstract: Context Cytological specimens allow minimally invasive, repeated sampling for diagnosis and molecular testing. However, cytology biobanking protocols have primarily been optimized for morphology, and the effects of preanalytical handling and storage on intracellular proteins remain unclear. Objective To assess how fixation, staining, storage temperature, and duration affect intracellular protein preservation in cytological specimens and to identify conditions suitable for in situ proximity ligation assay (isPLA). Design Human cancer cell lines were processed under different preanalytical conditions. Intracellular proteins and protein–protein interactions were assessed by isPLA, fluorescence microscopy, and quantitative image analysis. Slides were stored at room temperature or −20°C, with or without May–Grünwald–Giemsa (MGG) staining. Results Storage temperature had the greatest effect on protein signal. Unstained slides stored at −20°C retained strong signals for up to 24 months, whereas room temperature storage reduced detectable protein interactions by approximately 95%. MGG staining further reduced signal intensity during prolonged storage. Nevertheless, stained slides remained suitable for isPLA when staining, microscopic evaluation, destaining, and antibody labeling were completed on the same day. Prolonged room temperature storage of stained slides caused loss of antigenicity, increased autofluorescence, and false negative findings. Conclusions Low temperature storage and standardized preanalytical handling preserve intracellular protein signals in cytological specimens. These conditions support reliable single cell protein profiling of archived samples and their use in biomarker research and translational pathology.
Institutions: Uppsala University, Folktandvården Stockholms län AB
Language: English
Topic: Biotin and Related Studies
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
Funder: Cancerfonden
OpenAlex ID: W7220867566