abberior DNA-PAINT, FLASH kits

Introduction

The following protocol has been validated with abberior DNA-PAINT, FLASH dye conjugate. Depending on the model organism or target of interest, further optimization steps may be necessary. The following sections provide step-by-step instructions for: indirect immune FLASH-DNA-PAINT staining using anti-mouse, anti-rabbit, and anti-GFP secondary nanobodies (Indirect Immunostaining Kits), as well as direct immune FLASH-DNA-PAINT staining with a primary anti-GFP nanobody (Direct IF – GFP Kit).

The protocol is designed for adherent cells cultured on glass coverslips.

Storage

The product is shipped at room temperature. Upon arrival, store buffers at 4 °C and sdAb, Imager, and Adapter at −20 °C.

Immunofluorescence staining with abberior DNA-PAINT, FLASH for MINFLUX microscopy

The procedure below has been validated with abberior DNA-PAINT, FLASH dye conjugates, though further optimization may be needed for specific model organisms or targets. The following protocols describe indirect immune FLASH-DNA-PAINT staining using anti-mouse and anti-rabbit secondary nanobodies, as well as direct immune DNA-PAINT, FLASH staining using an anti-GFP nanobody, for adherent cells grown on glass coverslips.

Reagents, provided for indirect immunostaining kits

Secondary single domain antibodies (sdABs)

• FLASH sdAB Anti-rabbit + Docking Site A1, 100 µl (5 µM) (to be combined with adapter A1, imager IMG1) or FLASH sdAB Anti-mouse + Docking Site A2, 100 µl (5 µM) (to be combined with adapter A2, imager IMG2)
• Concentration: 5 µM Protein, 5 µM DNA (1 DNA strand per protein)
• Volume: 100 µL
• Storage: -20 °C
• Storage buffer: PBS, 50 % glycerol
• Recommended dilution: 1:200 – 1:500 (for optimal results the dilution needs to be optimized (depending on the target accessibility and expression level)

Adapter

• abberior DNA PAINT, FLASH adapter A1, 600 µl (20 µM) or abberior DNA PAINT, FLASH adapter anti-mouse A2, 600 µl (20 µM)
• Concentration: 20 µM in TE buffer (10 mM Tris, 1 mM EDTA, pH 8)
• Volume: 600 µL
• Storage: -20 °C (1 µM imager solutions are stable for multiple freeze-and-thaw cycles). Optional: Prepare 50 µL aliquots and store them at -20 °C. Working aliquots can be stored at 4 °C for short-term or -20 °C for long term.

Imager

• abberior DNA PAINT, FLASH imager IMG1, 300 µL (1 µM) or abberior DNA PAINT, FLASH imager IMG2, 300 µL (1 µM) carrying the respective fluorophore (580/660)
• Concentration: 1 µM in TE buffer (10 mM Tris, 1 mM EDTA, pH 8)
• Volume: 300 µL
• Storage: -20 °C (1 µM imager solutions are stable for multiple freeze-and-thaw cycles). Optional: Prepare 50 µL aliquots and store them at -20 °C. Working aliquots can be stored at 4 °C for short-term or -20 °C for long term.
  
  Note: Further dilutions should be prepared fresh before use. Low imager concentrations are not stable in plastic tubes.

Buffers

• abberior Antibody incubation buffer, 50 ml, stored at 2 – 8 °C
  
  Note: Blocking agents (BSA, serum, DNA etc.) are not provided and have to be added in the desired amounts before use.
• abberior Washing buffer (10×), 50 ml, store at 2 – 8 °C (to be diluted 1:10 in water before use)
• abberior Imaging buffer, 50 ml, store at 2 – 8 °C

Reagents, provided for the direct IF/GFP kit

Single domain antibodies (sdABs)

• FLASH sdAB Anti-GFP + Docking Site A3, 100 µl (5 µM)
• Concentration: 5 µM clone 1H1, 5 µM DNA (1 DNA strand per protein)
• Volume: 100 µl
• Storage buffer: PBS, 50 % glycerol
• Storage: -20 °C
• Recommended dilution: 1:200 – 1:500 (for optimal results the dilution needs to be optimized depending on the target accessibility and expression level)

Adapter

• abberior DNA PAINT, FLASH adapter A3, 600 µl (20 µM)
• Concentration: 20 µM in TE buffer (10 mM Tris, 1 mM EDTA, pH 8)
• Volume: 600 µl each
• Storage: -20 °C (1 µM imager solutions are stable for multiple freeze-and-thaw cycles). Optional: Prepare 50 µl aliquots and store them at -20 °C. Working aliquots can be stored at 4 °C for short-term or -20 °C for long term.

Imager

• abberior DNA PAINT, FLASH imager IMG3, 300 µL (1 µM) carrying the respective fluorophore (580/660)
• Concentration: 1 µM in TE buffer (10 mM Tris, 1 mM EDTA, pH 8)
• Volume: 300 µl each
• Storage: -20 °C (1 µM imager solutions are stable for multiple freeze-and-thaw cycles). Optional: Prepare 50 µl aliquots and store them at -20 °C. Working aliquots can be stored at 4 °C for short-term or -20 °C for long term
  
  Note: Further dilutions should be prepared fresh before use. Low imager concentrations are not stable in plastic tubes.

Buffers

• abberior Antibody incubation buffer, 50 ml, 30 ml, store at 2 – 8 °C
  
  Note: For longer-term storage, we recommend to store aliquots at -20 °C.
• abberior Washing buffer (10x), 50 ml (10×), 20 ml, store at 2 – 8 °C (to be diluted 1:10 in water before use)
• abberior Imaging buffer, 50 ml buffer, 50 ml, store at 2 – 8 °C

Required reagents (not provided)

• Fixative depending on cell-line, primary antibody or GFP construct (e.g.: 2 % – 8 % Formaldehyde in PBS (PFA), Methanol (abs.), or 4 % PFA + 0.02 % Glutaraldehyde in PBS)
• 1x Phosphate-buffered saline pH 7.4 (PBS)
• 1x PBS supplemented with 10 mM MgCl₂ (Magnesium chloride)
• BBI 150 nm gold colloid solution (SKU:EM.GC150), or Nanopartz Gold Nanoparticles 150 nm (A11-150-CIT-DIH-1-10)
• Two-component silicon glue

Staining procedure for cultured cells

1. Step 1 for indirect immunostaining kits
   Prepare the sample using a protocol optimized for your target and primary antibody staining. Wash with washing buffer (1×).

1. Step 1 for the direct IF/GFP kit::
   Perform the fixation of the sample using a protocol optimized for your GFP-tagged protein target.
2. Before the first use, add your blocking agents of choice to the abberior Antibody incubation buffer in the desired concentrations (Recommendation: 2% BSA).
3. Block cells in abberior Antibody incubation buffer for _~30 minutes.
4. Dilute sdABs in Antibody incubation buffer.
   
   Note: For most applications, a dilution of 1:200 of the sdAbs is sufficient. However, staining protocols may vary with cell type and application.
5. Incubate for 1 h at room temperature
6. Wash three times with washing buffer (1×).
7. Incubate the sample for 5 min with undiluted gold particle solution.
8. Wash three times with abberior Washing buffer (1×).
9. Incubate in abberior Washing buffer supplemented with 10 mM MgCl₂ for 5 minutes.
10. Wash once with abberior Imaging buffer.
11. Before imaging: Add imager strands and adapters diluted in imaging buffer. We recommend starting concentrations of 500 pM (1:2000) for imagers and 20 nM (1:1000) for adapters. However, the optimum imager concentration strongly depends on the target and labeling density. Thus, the imager concentration should be adjusted such that distinct single-molecule blinking events can be observed.
12. Samples can be mounted on cavity slides and sealed with two-component silicon glue for easy imaging and storage. Samples should be stored at 4 °C.

Abbreviations

MINFLUX Minimal fluorescence photon fluxes
GFP Green fluorescent protein
sdABs Single domain antibodies, nanobodies
PBS Phosphate-buffered saline
PFA Paraformaldehyde
MgCl₂ Magnesium chloride
min Minute
h Hour