Staining for GSD

Ground State Depletion (GSD) microscopy detects fluorophore positions independently of intensity or staining density.  As such, it is critical that proper blocking, fixing and staining methods be used in order to avoid off-target fluorescence.  Moreover, coverslips must be of #1.5 (0.17mm) thickness purchased from vendors who can provide coverslips with no fluorescent particulates.  That, or coverslips must be rigorously cleaned (e.g. Piranha cleaning).  At this time the best coverslips appear to be EMS #72222-01.  As of this writing, coverslips from VWR and Fisher are not GSD-compatible.

Note: careful selection of secondary antibody fluorophores is key to success in GSD.  Make sure you are using established GSD-compatible fluorophores.

Protocol - Fixation

Careful fixation is required to stabilize cell structures while preserving antigenicity and fluorescent protein activity. The following fixation protocol generally works well:

1. Fix cells in 4% PFA in PBS.
   • If cytoskeletal structure is critical, fix for 10min at 10-12C, followed by 20 min at room temperature
   • otherwise, fix for 20 min at room temperature
     
2. Wash 3X PBS, 30 sec/wash.
3. Continue staining as follows:
   • If cells were transfected with fluorescent proteins and no other staining is required, store cells in blocking buffer until imaging.
   • If intracellular structures are to be immunostained, follow the instructions for intracellular staining. Use this protocol for both intracellular and mixed intracellular/extracellular staining.
   • If only extracellular structures are to be immunostained, follow the instructions for extracellular staining.
     

Protocol - Intracellular Staining

Use this protocol when immunostaining intracellular structures. Use this protocol if simultaneously staining intracellular and extracellular structures.

1. Permeabilize and block using permibilization buffer, 1hr at RT.
   • During permeabilization, prepare primary and secondary antibodies in permeabilization buffer. Antibody concentrations are generally the same as those used for conventional immunofluorescent microscopy.
2. Stain with primary antibody, room temperature, for 1 to 1.5 hours:
   • Use hanging drop method:
     • Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm. A highly elevated drop should form.
     • "Float" coverslip, cell-side-down, on drop.
     • Cover with foil or box and leave undisturbed (KEEP LEVEL).
3. Wash 3X 15min with blocking buffer.
4. Add secondary antibody(s) in permeabilization buffer. Conventional staining or the hanging drop method can be used
   • Secondary staining should be for 1-2 hours, room temperature.
5. Wash 3X 15 min in PBS (no BSA).
6. Post-fix with 2% PFA in PBS, 20 min at room temperature
7. Wash 3X 30 sec with PBS (no BSA)
   

Cells should be maintained in PBS until imaging. For imaging cells need to be either mounted in a depression slide using a GSD-appropriate media (e.g. PBS + 50mM cystamine), or coated in PVA.

Protocol - Extracellular Staining

This protocol should be used only for staining extracellular structures. If staining both extracellular and intracellular structures, pool the antibodies and follow the intracellular staining protocol, above.

1. Block the sample for 1 to 1.5 hours with 5% BSA in PBS
2. Stain with primary antibody, room temperature, for 1 to 1.5 hours:
   • Use hanging drop method:
     • Put 100ul of antibody(s) in permeabilization buffer on a piece of parafilm. A highly elevated drop should form.
     • "Float" coverslip, cell-side-down, on drop.
     • Cover with foil or box and leave undisturbed (KEEP LEVEL).
3. Wash 3X 15 min with PBS + 5% BSA.
4. Add secondary antibody(s) in permeabilization buffer. Conventional staining or the hanging drop method can be used
   • Secondary staining should be for 1-2 hours, room temperature.
5. Wash 3X 15 min in PBS (no BSA).
6. Post-fix with 2% PFA in PBS, 20 min at room temperature
7. Wash 3X 30 sec with PBS + 5% BSA.
   

Fluorophore & Fluorescent Protein Selection

Fluorophore	Excitation	Emission	Notes
Cy3	550	570	Slow to deplete, but blinks forever. Orange/Red emission.
Dylight 649	650	670	Best tested fluorophore to-date. Far-Red emission.
YFP	514	527	Only confirmed GSD-compatible fluorescent protein at this time. Cannot be combined with orange/red emitting fluorophores
Alexa 488	488	505	Only good green fluorophore identified to date. Slow to deplete but blinks well.
Alexa 555	555	565	Equivalent in performance to Cy3, perhaps with slightly better photostability
Alexa 647	647	671	Similar to Dylight 649, but faster depleating. Blinks forever.

Sample Preparation Buffers

4% PFA

1 mL of fixative is required for each well of a 12-well plate, or 1.5 mL per well of a 12-well plate. For 4 mL of PFA mix:

• 400 ul of 10x PBS
• 1 ml of 16% PFA
• 2.6 ml of ddH2O

2% PFA

1 mL of fixative is required for each well of a 12-well plate, or 1.5 mL per well of a 12-well plate. For 4 mL of PFA mix:

• 400 ul of 10x PBS
• 0.5 ml of 16% PFA
• 3.1 ml of ddH2O

Blocking Buffer

Blocking buffer is 5% bovine serum albumin (BSA) in PBS. For 10ml mix 10ml PBS with 0.5g of BSA.

Permeabilization Buffer:

Permeabilization buffer is Blocking Buffer + 0.1% Triton X-100. For 10ml mix 10ml blocking buffer with 10ul Triton X-100.

GSD Imaging Buffers

GSD requires specialized buffers to prevent the triplet state (depleted state) of the fluorophores from being oxidized. This can be achieved by providing a reducing agent (buffer 1) to protect the triplet state, depletion of oxygen in the sample (buffer 2), or a combination of the two (buffer 3). A new buffer has recently been published, but we are still assessing its effectiveness (buffers 4 & 5).

GSD Buffer 1 (MEA):

This is the standard buffer that works for most experiments. It is comprised of PBS, pH 7.4, plus 10mM to 100mM MEA (β-Mercaptoethylamine [AKA Cystamine], Sigma-Aldrich #30070). For 10ml of 50mM/100mM MEA:

• 75mg/150mg of MEA to 10ml of PBS
• MEA will cause the pH of the PBS to change. Return pH to 7.4
• Filter with a 0.22um filter to remove particulates
• Aliquote 1ml into 1.5ml tubes, freeze at -20C
  

Use a fresh aliquot each day, do not re-freeze buffer. Frozen aliquots are only good for 1-2 months, after which the buffer must be replaced. Lower MEA concentrations will last for shorter periods of time than higher concentrations.

GSD Buffer 2 (Glucose-Oxidase):

This binary buffer scavenges oxygen from the media. It may work better for some fluorescent proteins than buffer 1, but tends to have higher background fluorescence.

Prepare:

• 10x enzyme mix: 5 mg/mL glucose oxidase + 400 μg/mL catalase in PBS + 15% glycerol, pH 7.4. This can be aliquoted and frozen at -20C until needed.
• 11% glucose in PBS, pH 7.4.
  

Mounting:

• For each coverslip, immediately before mounting, pre-mix 10ul of enzyme mix with 90ul of glucose/PBS.
• Mount coverslips as quickly as possible.

GSD Buffer 3 (Combination):

This is a combination of buffers 1 & 2. While used in the literature, it is not clear if this offers any additional advantages over either buffer 1 or buffer 2 alone. This buffer may work better for tissue sections.

Preparation:

• Prepare buffer 2 as per usual, but to the glucose/PBS mix add 50mM MEA, and re-pH the solution to 7.4.
• Buffer is mixed and mounted as per buffer 2.

GSD Buffer 4 (Sulfite Buffer):

This is a newer buffer formulation designed for dSTORM that has a longer shelf-life and superior imaging characteristics to buffer 1. It requires optimization for specific fluorophores in GSDM and usually does not work "out of the box".

• 10 mM MEA (1.52 mg/mL, 15.2 mg per 10 mL)
• 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
• PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.

GSD Buffer 4 (AlexaFluor Optimized Buffer):

This is GSD Buffer 4 optimized to work well with Alexa Fluor 488, 555, and 647. It also works well with Cy3. This is the best buffer for these fluorophores that we have tested to-date.

• 25 mM MEA (3.75 mg/mL, 37.5 mg per 10 mL)
• 20 mM Sodium sulfite (2.52 mg/mL, 25.2 mg per 10 mL)
• PBS

Dissolve MEA and Sodium sulfite into PBS. Adjust pH back to pH 7.4 and aliquote. Freeze until needed, should last 2-3 months frozen. Do not reuse aliquots.