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storm_sample_preparation

STORM Sample Preparation

Example of Dye Preparation and Antibody Labeling Primary or secondary antibody of your preference

Repofter Dye

  • AlexaFluor 647 Carboxylic acid, Succinimidyl ester 1 mg #420006 [Invitrogen]

Activator Dyes

  • Alexa Fluor 405 Carboxylic acid, Succinimidyl ester 1 mg #A30OOO [Invitrogen]
  • Cy2 Reactive Dye Pack 5 vials #PA22000 [GE Healthcare]
  • Cy3 Mono-reactive Dye Pack 5 vials #P423001 [GE Healthcare]

Other Reagents

  • Anhydrous Dimethyl Sulfoxide
  • PBS

Equipment

  • NAP-5 columns #17-0853-02 [GE Healthcare]
  • Evaporator
  • Shaking platform
  • Nanodrop (protein-probe),or UV/Visible absorption spectrophotometer

Dye preparation Dissolve the Alexa 647 dye according to the manufacturers recommendations in anhydrous DMSO and aliquot into tubes for a final 0.02 mg amount of dye per tube. For the Cy dyes, dissolve one dye pack into a sufficient amount of anhydrous DMSO to allow distribution into 10 new aliquots. Evaporate all DMSO Store the aliquots in -20°C in the dark.

Labeling Dissolve one activator (Alexa405, Cy2, or Cy3) aliquot and one reporter aliquot in in 10 pl DMSO respectively.

Mix:
50 pl antibody (I.25 mg/ml in PBS)
6 pl <chem>NaHCO3</chem>
1.5 pl Cy3
0.6 pl Alexa647

Incubate 30 minutes on a shaking platform in room temperature

Elution Equlibrate Nap-S gel filtration columns (one per labeling reaction) with three volumes of PBS Bring up the reaction volume to 200 pl with pBS Add the whole reaction volume to the center of the column and allow the sample to enter the column. After the last drip, immediately add 550 pl pBS to wash Add 300 µl PBS and collect the elutent. Measure the absorbance of the labeled antibody in a Nanodrop (protein-probe module) or in an UV/Vis spectrophotometer Antibody can be stored at 4°C for up to 6 months 

Ratio calculations

The labeling ratio of the antibody must be determined for each individual application. In general, to make sure that all antibodies has at least one activator, it is recommended to have more than one activator dye molecule per IgG. Reporter dye molecules should be less than one per IgG. To many reporters can increase non-specific activation and will also add size to the structure of interest. If a labeled primary antibody is used, signal amplifi- cation is lost and higher labeling ratios maybe helpful. Start with a 3-5 times higher concentration of activator than repofter. The dye concen- trations needed to achieve ideal labeling ratios needs to be determined empiristically. So once the dye to antibody ratio has been determined, the labeling may need to be repeated with adjusted dye concentrations.

The recommended labeling ratio can be used as a guideline, but needs to be tested for each individual application

Formulas

Antibody concentration [IgG] = A280 - (Adye1 x CF280 dye1) - (Adye2 x CF280 dye2) / ƐIgG Dye concentration [Dye] = (Absdye x dilution) / Ɛdye

CF280 = Correction factor for 280 nm (labeling dye) Ɛ = Molar extinction coefficient (a measurement of how strongly a chemical species absorbs light at a given wavelength)  Labeling Ratio = [Dye] / [IgG]

Example on ratio calculation

Measure the absorbance of the antibody at: 28O nm (protein, IgG) 55O nm (max. absorbance Cy3) 650 nm (max. absorbance Alexa 647)

Dyeƴ (nm)AbsƐCF280
IgG 2800.251210 000
Cy35500.333150 0000.08
Alexa 6476500.156239 0000.03

IgGI = (0.251 - (0.333xO.OB) - (0.156xO.O3)) | 2f-O OOO = 1.O5x1O-6M = l.O5 pM lCy3l = O.333 | l5O OOO = 2.22 x 10-6 M = 2.22ltlt 1A6477 = O.156 | 239 OOO= 6.5 x l.O-7 M = O.65 UM I f

Labeling Ratio

Cy3 I I.gG = 2.22 yln I L.OS FM = z.LL A647 | IgG = O,65 yUl I 1.O5 HM = 0.62 150 000 0.08 239 000 0.03 { I

STORM Sample Preparation

Example staining protocol

Staining protocols needs to adjusted and optimized for each application and target. Follow a staining protocol that works for your cells as far as it is possible as fixation, permeabili- zation, and labeling conditions that work for one target can be incompatible with another target.

Tubulin staining of HeLa cells

Material and reagents LabTek II 8-well chambered coverglass (Nunc) PBS PFA 4% in PBS 0.1 % Glycine Blocking solution: to/o BSA + 5olo donkey serum Triton-X-100 Mouse anti-p-tubulin diluted in 1 % BSA Labeled secondary antibody (donkey anti- mouse IgG) diluted in 1 o/o BSA Nunc Lab Tek II chambered cover glass

Optional Chambered coverglasses may have fluorescent contaminants that can give background. To remove them, clean Lab-Tek II coverglasses before plating the cells. They can be sonicated for 20 minutes in 1 M potassium hydroxide. Rinse with MQ water and sterilize under UV-light for more than 30 minutes.

Protocol (all incubations in room temperature and on a rocking platform)

  • Plate cells (about 12 000 / chamber) on chambered coverglass and incubate until a confluency of 6O-70% is reached
  • Remove from incubator and rinse thoroghly with PBS.
  • Fix with 200 Ul 4 o/o PFA
  • Wash 3 times in PBS
  • Permeabilize in 0.5 o/o Triton X-100 for 3 minutes
  • Wash 3 times in PBS
  • Block free alde
  • Wash 3 times in PBS
  • Block with 1 o/o BSA + 5 o/o donkeyserum for 30 minutes
  • Aspirate the blocking buffer and replace it with the primary antibody (diluted i nIo/o BSA)
  • Incubate with primary antibody for 60 minutes
  • Wash 3 times in PBS
  • Incubate with labeled secondary antibody for 30 minutes
  • Wash 3 times in PBS
  • Postfixation in 4o/o PFAfor 7 minutes
  • Wash 3 times in PBS
  • Store in PBS at 40 C. For long time storage, store in PBS + 20mM NaNr.

I, *' 0.1 M Glycine for 10 minutes i ¡o w-. R*t“

Buffers for STORM imaging

Immediately before STORM imaging is stafted, a specific buffer is added to the sample. The buffer contain p-mercaptoethanol or cysteamine (MEA) as the organic fluorophores used require a thiol in the buffer to photoswitch effectively. What is used depends on the application. For single color imaging, p-mercaptoethanol is recomended as it generates brigth, long-lived single molecule photoswitching events. Cysteamine reduces the observed number of photons slightly but also reduces the non-specific blinking making it ideal for multicolor STORM imaging. Additionally, glucose oxidase and catalase is added and used as a oxygen scavenging system for reduction of photoblaching.

Stock buffers prepared before STORM imaging

Dilution Buffer 10 mM Tris, pH 8.0 50 mM NaCl Oxygen Scavenger (GLOX) t4 mg Glucose Oxidase 200 pl Dilution Buffer 50 pl Catalase 20 mg/ml Dissolve glucose oxidase in PBS, add catala speed for 1 minute. Use only the yellow supernatant for the buffer. Can be stored at 40 C for up to 2 weeks.

MEA 77 mg Mea(Cysteamine) dissolve in 1 ml 360 mM HCL Can be stored at 40 C for up to 1 month

Fixed Cell Imaging Buffer Base tlo/o Glucose (m/v) 50 mM Tris, pH 8.0 10 mM NaCl Can be stored at room temperature for up to 6 months

Live Cell Imaging Buffer Base 10 ml cell medium of choise, ex DMEM, no phenol red 750 pl HEPES, pH adjusted to 8.0 400 pl 50o/o Glucose Can be stored at 40 C for up to 2 weeks mix well, Centrifuge at maximum

Buffers for STORM imaging

STORM imaging buffers are quickly degraded in an open environment due to acidification of the buffer as the glucose oxidase/catalase system will make th pH to drop over time. The switching behaviour of the fluorochrome is sensitive for the pH in the solution. It should be aroundT or higher. Therefore, they must be prepared and added to the sample immediately before imaging starts. Usually they last for 30-60 minutes but a well sealed sample can extend the imaging for up to several hours. After imaging, replace the imaging buffer with PBS ¡f you want to keep the samples.

Fixed Cell STORM Imaging Buffer (prepare just before imaging)

Volume for 1 well in a Lab-Tek II chambered coverglass. Prepare on ice.

690 Ul Fixed Cell Imaging Buffer Base 7 pl p-mercaptoethanol 7 pl GLOX 650 Ul Fixed Cell Imaging Buffer Base 70pl lMMEA 7 pl GLOX

Live Cell STORM Imaging Buffer (prepare iust before imaging)

Volume for 1 well in a Lab-Tek II chambered coverglass. Prepare on ice

690 pl Live Cell Imaging Buffer Base 3.5 pl p-mercaptoethanol 7 pl GLOX 690 Ul Live Cell Imaging Buffer Base 4.2¡tl 1M MEA 7 ¡rl GLOX

storm_sample_preparation.txt · Last modified: 2020/02/27 18:22 (external edit)