Question: Is a cDNA footprint signal in my Tissue Optimization experiment negative control with IF staining expected?
Answer: The negative control during the Tissue Optimization (TO) experiment may show minimal fluorescence signal. It serves as a baseline for determining "background" fluorescence, allowing customers to reduce the laser power or gain such that the negative control fluorescence is no longer observed. This, in turn, allows for customers to visualize fluorescence only attributed to the tissue permeabilization time course during the TO assay.
Observing low fluorescence is expected with immunofluorescence (IF) stained tissue sections. The blocking, staining, and washing buffers used in the IF protocol contain a small amount of surfactant that enables removal of non-specific antibody binding as well as permeabilization of the tissue in order to facilitate intracellular protein staining. These conditions, in turn, also often result in the release of a small amount of mRNA.
This is demonstrated in the TO experiment images shown below where 10 µm tissue sections from mouse brain were stained with DAPI (blue) using the Methanol Fixation, Immunofluorescence Staining & Imaging Demonstrated Protocol. The DAPI channel (left) and the TRIC channel (right) are shown as separate images. The negative (No perm) control is outlined with a white box in both images.
Please note: This experiment excluded a positive control only for demonstration purposes.