Immunofluorescence on FFPE tissue sections from inflamed and non-inflamed human gut v1

Analysis of immunofluorescence is commonly confounded by autofluorescence and non-specific staining in the colorectum, particularly within highly inflamed samples. There is no consensus on how to definitively prevent this, and study protocols are variable. Our laboratory identified strong immunofluorescence within negative control samples stained for numerous target antigens, including CD3. Following methodical attempts to ensure there was no reagent contamination, we concluded that this was autofluorescence/ non-specific staining. Significant optimisation was subsequently performed. In this protocol, we report our optimised methodology for immunofluorescence experiments on formalin fixed paraffin embedded (FFPE) tissue sections, stained for identification of CD3+ lymphocytes. We hope this protocol acts as a template to help other laboratories optimise their own immunofluorescence protocols for similar antibodies.