STORM Immunofluorescence Protocol

  1. Solutions
    • Fixative: 3% paraformaldehyde + 0.1% glutaraldehyde in PBS)
    • Fixative Quench: 0.1% Sodium borohydride (NaBH4) in PBS
    • Blocking Buffer (BB): 3% BSA, 0.2% Triton X-100 in PBS
    • Washing Buffer (WB): 0.2% BSA, 0.1% Triton X-100 in PBS
    • Imaging buffers:
      • 10 % Glucose solution in PBS
      • Oxygen scavenger system:
        • Final: 0.5 mg/ml glucose oxidase (Sigma G2133). Stock is 100 x concentrated in freezer
        • Final: 40 μg/mL catalase (Sigma C40). Stock is 1000 x concentrated in freezer
        • 143 mM of β-mercaptoethanol (βME) at pH 8.0 to allow AF647 dye photoswitching OR
        • 10 mM Mercaptoethylamine (MEA or Cysteamine, Sigma 30070-10G)
  2. Immununostaining Protocol
    • Grow cells on #1.5 coverslip that can be assembled into growth chamber or on the bottom of a 35mm glass bottomed dish.
    • Wash coverslip with 500 μl PBS.
    • Fix cells with 3% formaldehyde + 0.1% glutaraldehyde (diluted in PBS) for 10 minutes at RT.
    • Wash 3 times with PBS
    • Immediately before use, prepare 0.1% (w/v) NaBH4 in PBS.
    • Reduce remaining PFA/Glut with 0.1% sodium borohydride for 7 minutes at RT.
    • Wash coverslip three times with 500 μl PBS
    • Block and permeabilize cells in blocking buffer (BB) for 30 minutes at room temperature.
    • Add primary antibody and incubate for 30 mins at RT (dilutions in BB). Below are example of antibodies used in Zhuang X’s papers:
      • Microtubules: 1:10 mouse anti beta-tubulin (Sigma T5201)
      • Clathrin: 1:100 rabbit anti clathrin heavy chain (abcam ab21679, 1 mg/ml)
      • In STORM, labelling density becomes important, so as a starting point, use primary antibodies at 10X more concentrated than if you were going to image in a widefield epifluorescence or confocal microscope
    • Wash 5X with 200 μl washing buffer (WB) for 10 min/wash. Check box to keep track of washes □ 1 □ 2 □ 3. □ 4 □ 5
    • Add secondary antibody diluted in BB and incubate for 30 min at RT. Protect from light !! Please see below for example of secondary antibody dilutions:
      • 1:100 AF647 donkey anti mouse
      • 1:100 AF488 donkey anti rabbit (or ATTO 488)
    • Wash with WB 3 times for 10 min. □ 1 □ 2 □ 3
    • Post-fix for 10 minutes at RT with fixation buffer (as above – no sodium borohydride step). This seems to be important to fix antibodies in place to increase resolution during imaging.
    • Wash 3 times with PBS
    • For long term storage, keep at 4˚C and add 20 mM sodium azide in PBS
  3. Imaging
    • Prepare imaging buffer fresh by diluting a 20 % glucose/PBS stock solution 2 fold in PBS and adding:
    • Add 1/100 of Glucose Oxidase stock
    • Add 1/1000 of Catalase stock
    • Add 1/100 of 2-mercaptoethanol
    • Use once for up to a hour in a well-sealed sample
    • Change the solution from time to time to restore the photoswitching capabilities of the dyes