, Prepare the solution of primary antibodies using anti-vimentin and anti-tubulin with a 1:500 dilution in the blocking solution

, Put 100 µL drops of diluted primary antibodies on a paraffin film layer and place the coverslips on top of them with cells facing downward

, Incubate cells with primary antibodies for 2 h. Put the coverslips back in a 12-well plate filled with PBS. Rinse thrice for 5 min each time in PBS at room temperature on an orbital shaker

, secondary antibodies solution using anti-mouse Alexa647 and anti-rat Alexa555 at a dilution of a 1:1, 000 in the blocking solution. Incubate cells with secondary antibodies for 1 h at room temperature

, Put the coverslip back in a 12-well plate filled with PBS. Rinse thrice for 5 min each time in PBS at room temperature on an orbital shaker. Remove the PBS and add 0.5 mL of PBS mixed

, Put the wells on an orbital shaker for 30 min. Rinse with PBS. Remove the PBS and incubate the cells for 5 min with a 4% PFA solution in PBS. Rinse thrice in PBS. Note: Fixed cells can be stored for a couple of weeks in PBS at 4°C

, Open your STORM raw data and use the Image processing tab. Select the PALM method and then PALM again

, Press Apply to start the reconstruction using a peak mask size of 9 and a peak intensity to noise of 6 (or the parameters you choose for the image)

, Select the rectangle Graphics tool and draw a rectangle on the raw image around a single molecule present on the glass surface. There are usually a few single molecules there

, Press Apply again, and only the molecules present inside the rectangle region will be analyzed

, In the PAL-Statistics tool, select Plot type: histogram, and Histogram Source: X position or Y position, to visualize the position histograms

, Using a software of data analysis, create histograms of X and Y positions (Figure 1D)

, Fit the distributions by a Gaussian and save the ? X and ? Y values. The localization precision ? SMLM is estimated by

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