Fig. 1: Cassini: evaluating RNA and protein detection performance.

a Overview of Cassini. b Effect of Cassini on conventional immunostaining and signal retention after to 2nd fixation. Microscope images display immunostaining prior to Cassini processing, with the white outlines indicating the regions selected for signal retention quantification shown in bar charts below. Dot plots represent biological triplicates for each condition. c Buffer optimization for conjugated antibodies. The microscope images show a zoom area of the CA1 hippocampal field. The white lines indicate the areas measured for signal intensity, which are plotted on the left. All conjugated immunostaining utilized the same lookup table (LUT) settings, while the conventional immunostaining images were enhanced to match the intensity levels of the conjugated staining. d Comparison of conventional and oligo conjugated immunostaining after Cassini run. The DAPI signal is shown in blue in both conjugated and regular antibody images, but not in the merged image to facilitate visualization. The lookup table of conventional and conjugated immunostaining have been adjusted to match the intensity. e Violin plot of foci area of HCR and Cassini. Bars in the violin plots indicate the standard deviation of the data. Violin plots were generated from selected areas with low foci density, aggregated from biological triplicates for each gene. f Comparison of foci density between Cassini and HCR. Density is the average number of foci around each dot in a 10 μm radius. At the top left is a schematic of the density calculation, and at the bottom left are the different genes and areas that were considered. Dot plots represent biological triplicates for each condition (except for Cux2 Cassini series with only two replicates). Asterisks (*) indicate p < 0.05 without correction for multiple testing (one-tailed Student’s t test with df = 4). Prox1, p = 0.0152; Slc17a7-CA-Lateral, p = 0.048; and Slc17a7-CA-Medial, p = 0.038. g Cassini/HCR ratios along the Amigo2 gradient. The sliding window was 10% of the total length with an increment of 2%. The blue line indicates the average of all possible Cassini/HCR ratios (points) from triplicates experiments calculated for each increment. Note: Cassini data in e–g do not involve immunostaining and are limited to RNA detection.