| Fixation Protocol . | IF SNR Rab5 . | IF SNR Rab7 . | IF SNR EEA1 . | IF SNR APPL1 . | EM Morphology . | Figure . |
|---|---|---|---|---|---|---|
| 30-min FA 4% | 2.09 ± 0.05 | 1.61 ± 0.03 | 2.00 ± 0.06 | 2.30 ± 0.11 | – | Fig. S1 A |
| 1-h FA 4% | 1.72 ± 0.15 | 1.66 ± 0.07 | 1.76 ± 0.08 | 1.85 ± 0.06 | +/− | Fig. S1 B |
| 2-h FA 4% | 1.62 ± 0.34 | 1.51 ± 0.03 | 1.42 ± 0.04 | 1.63 ± 0.03 | +/− | Fig. S1 C |
| 1-h FA 4% → ON FA 0.6% | 1.44 ± 0.07 | 1.48 ± 0.06 | 1.41 ± 0.04 | 1.66 ± 0.06 | + | Fig. S1 D |
| ON FA 4% | 1.43 ± 0.13 | 1.51 ± 0.02 | 1.24 ± 0.02 | 1.31 ± 0.02 | + | Fig. S2 A |
| 30-min FA 4% + GA 0.2% | 1.66 ± 0.06 | 1.36 ± 0.03 | 1.20 ± 0.04 | 1.28 ± 0.08 | ++ | Fig. S2 B |
| 2-h FA 4% + GA 0.2% | 1.70 ± 0.02 | 1.41 ± 0.03 | 1.34 ± 0.05 | 1.39 ± 0.04 | ++ | Fig. S2 C |
| ON FA 4% + GA 0.2% | 1.64 ± 0.04 | 1.36 ± 0.02 | 1.25 ± 0.01 | 1.30 ± 0.02 | ++ | Fig. S2 D |
| Fixation Protocol . | IF SNR Rab5 . | IF SNR Rab7 . | IF SNR EEA1 . | IF SNR APPL1 . | EM Morphology . | Figure . |
|---|---|---|---|---|---|---|
| 30-min FA 4% | 2.09 ± 0.05 | 1.61 ± 0.03 | 2.00 ± 0.06 | 2.30 ± 0.11 | – | Fig. S1 A |
| 1-h FA 4% | 1.72 ± 0.15 | 1.66 ± 0.07 | 1.76 ± 0.08 | 1.85 ± 0.06 | +/− | Fig. S1 B |
| 2-h FA 4% | 1.62 ± 0.34 | 1.51 ± 0.03 | 1.42 ± 0.04 | 1.63 ± 0.03 | +/− | Fig. S1 C |
| 1-h FA 4% → ON FA 0.6% | 1.44 ± 0.07 | 1.48 ± 0.06 | 1.41 ± 0.04 | 1.66 ± 0.06 | + | Fig. S1 D |
| ON FA 4% | 1.43 ± 0.13 | 1.51 ± 0.02 | 1.24 ± 0.02 | 1.31 ± 0.02 | + | Fig. S2 A |
| 30-min FA 4% + GA 0.2% | 1.66 ± 0.06 | 1.36 ± 0.03 | 1.20 ± 0.04 | 1.28 ± 0.08 | ++ | Fig. S2 B |
| 2-h FA 4% + GA 0.2% | 1.70 ± 0.02 | 1.41 ± 0.03 | 1.34 ± 0.05 | 1.39 ± 0.04 | ++ | Fig. S2 C |
| ON FA 4% + GA 0.2% | 1.64 ± 0.04 | 1.36 ± 0.02 | 1.25 ± 0.01 | 1.30 ± 0.02 | ++ | Fig. S2 D |
90-nm cryosections of HeLa cells fixed according to the indicated protocols were fluorescently labeled and imaged using fixed settings. The SNR was calculated as mean intensity value of the 0.5% brightest pixels divided by the mean intensity value of the reverse selection. Five fields-of-view were averaged for each measurement. Most SNRs significantly declined upon >30-min FA fixation or addition of GA. Morphological quality of EM images was based on blind ranking by six experienced electron microscopists. Based on these measurements, 1-h fixation with 4% FA was chosen as best fixative for CLEM.