Supplementary Figure 2: Validation of commercial antibodies targeting cell-type markers in MAP-processed tissues. | Nature Biotechnology

Supplementary Figure 2: Validation of commercial antibodies targeting cell-type markers in MAP-processed tissues.

From: Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues

Supplementary Figure 2

Fluorescence images from various commercial antibodies targeting cell-type markers tested in both MAP and control samples. Control and MAP samples were sectioned to 100-μm thickness and then stained after denaturation. Primary incubation was performed for 12 h at 37°C with gentle shaking followed by a two-step wash of 2 h each in PBST. Secondary incubation was performed for 6 h at 37°C, followed by a 2-h wash in PBST, 30 min in 1:50,000 DAPI solution, then another 2-h wash in PBST. To determine specificity, antibodies targeting the same antigen were tested simultaneously in a single tissue using separate color channels when possible. Images were acquired with our Olympus confocal microscope with the following settings: 550 HV, 10 μs pixel−1 dwell time, 1,024 × 1,024 resolution, 0% offset, and laser power sufficient to nearly saturate signals. A 20×, 0.95 NA water-immersion objective was used. MP, Millipore; BL, BioLegend, CST, Cell Signaling Technology. Scale bars, 20 μm.

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