Fig 1: Increased Immunolabeling Efficiency in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval(A and B) Post-embedding fluorescent labeling for SNAP25 on 200-nm-thin sections obtained from rat cerebellar cortex embedded into three different types of resin without (A) or with 2 min of etching with Na-ethanolate (B). All images were taken under the same illumination and detection conditions.(C) Munc13-1 immunolabeling of the stratum radiatum in epoxy-resin-embedded mouse hippocampal CA1 area after etching and retrieval. Reactions were carried out on sections with different thicknesses.(D) Normalized mean fluorescent signal intensity of PSD95, Munc13-1, and vGluT1 show a tight linear correlation with the section thickness. Symbols for each protein are the mean ± SD from 3 reactions in 3 mice. The reaction strengths were normalized to those obtained in the 200-nm-thick sections.(E) Immunofluorescence for vGluT1 in the stratum radiatum of the CA1 area (left image). Circular ROIs were placed over fluorescent clusters (ROI #1–#4) and over the unlabeled neuropil to determine the specific and background (bg) labeling, respectively. The reaction was followed by an elution step with 1% SDS (middle image) and a step when only the appropriate sAb (Alexa647 coupled donkey anti-rabbit) was applied (right image). All images were taken with the same acquisition settings and are shown with the same look-up table.(F) The integrated fluorescence (background-subtracted mean) is plotted for the 1st labeling round, the elution, and the sAb relabeling steps. When the sAb was reapplied, the mean fluorescence did not increase significantly (1.4% ± 0.5% versus 2.3% ± 1.1% of the 1st labeling round, n = 7, p = 0.99; while both significantly differed from the 1st labeling round, p < 0.0002, Kruskal-Wallis with Tukey honestly significant difference [HSD] post hoc test). Measurements for 7 proteins are shown, and symbols for each protein are the mean ± SD from 3 reactions in 3 mice from 20–50 ROIs per reaction.(G) Mean normalized PSD95 fluorescence is shown in 4 consecutive labeling rounds in epoxy-resin-embedded tissue obtained from 3 mice (open circles, 60, 69, and 73 ROIs in 3 reactions). The mean ± SD are shown with filled circles.(H) Same as in (G), but labeling for 4 proteins are shown (all represent the mean ± SD of 3 mice; 20–73 ROIs in each reaction).(I) The background-subtracted fluorescence of each ROI (same as in H) was calculated and normalized for the mean of each labeling round. Then, the ratio of the 2nd and 1st rounds was calculated for each ROI, and the mean and the coefficient of variation (CV) was calculated of these ratios. This was repeated for the 3rd and 2nd rounds and for the 4th and 3rd rounds. The symbols represent the mean ± SD from 3 reactions (3 mice).(J) The ratios of the background-subtracted fluorescent signal for vGluT1 and PSD95 and for Munc13-1 and PSD95 were calculated across 4 labeling rounds (27–30 ROIs in each experiment in 3 mice). Then, the ratios of the 2nd and 1st, 3rd and 2nd, and 4th and 3rd rounds were calculated. The round-to-round variability (CV) was then calculated and plotted.Open symbols represent the CV values of the 3 reactions in 3 mice, whereas filled symbols indicate the mean ± SD.Abbreviations are as follows: ml, molecular layer; pcl, Purkinje cell layer; gcl, granular layer.