catalase (ADC) supplement and then sterile-filter, or first autoclave at 121
C for 10 min, allow to cool to room temperature,
and then aseptically add 100 mL of ADC supplement. Store at
4
C.
3. 1Â phosphate-buffered saline (1Â PBS): In a clean 2 L
graduated beaker, add 8 g NaCl, 0.2 g KCl, 1.44 g
Na 2 HPO 4 , and 0.24 g KH 2 PO 4 . Add 800 mL of ultrapure
water and stir to dissolve. Adjust the pH to 7.4 using HCl. Add
ultrapure water to a final volume of 1 L. Sterile-filter and store
at 4
C.
4. 0.5% bovine serum albumin in 1Â PBS (PBSB): Dissolve 0.5 g
of bovine serum albumin in 100 mL of 1Â PBS, sterile-filter,
and store at 4
C.
5. 4% formaldehyde in PBS: Protect from light and store at 4
C.
2.4 CopperCatalyzed Azide–
Alkyne Cycloaddition
(CuAAC) Reaction
1. 1 mM Az-488: Dissolve 1.0 mg of carboxyrhodamine
110 Azide (Click Chemistry Tools) in 1.7 mL of DMSO.
Vortex briefly to dissolve and store at À20
C (see Note 3).
2. 1 mM Alk-488: Dissolve 1.0 mg of carboxyrhodamine
110 Alkyne (Click Chemistry Tools) in 1.7 mL of DMSO.
Vortex briefly to dissolve and store at À20
C (see Note 3).
3. 60 mM sodium ascorbate: Dissolve11.9 mg of (+)-sodium Lascorbate (Millipore Sigma) in 1.0 mL of ultrapure water.
Vortex briefly to dissolve. Prepare fresh and use immediately
to avoid oxidation.
4. 6.4 mM TBTA: Dissolve 3.4 mg of tris(benzyltriazolylmethyl)amine (TBTA) ligand (Click Chemistry Tools) in 1.0 mL of tBuOH/DMSO (3:1 v/v). Vortex briefly to dissolve and store
at À20
C (see Note 4).
5. 50 mM Copper sulfate: Dissolve12.5 mg of copper(II) sulfate
pentahydrate (Millipore Sigma) in 1.0 mL of ultrapure water.
Vortex briefly to dissolve and store at 4
C.
2.5 Strain-Promoted
Azide–Alkyne
Cycloaddition (SPAAC)
and Tetrazine
Reactions
1. 1 mM Cy3 DBCO: Dissolve 1.0 mg of Cy3 DBCO (Click
Chemistry Tools) in 1.0 mL of DMSO. Vortex briefly to dissolve and store at À20
C (see Note 3).
2. 1 mM Cy3 tetrazine: Dissolve 1.0 mg of Cy3 tetrazine (Click
Chemistry Tools) in 1.1 mL of DMSO. Vortex briefly to dissolve and store at À20
C (see Note 3).
2.6 Fluorescence
Microscopy
1. Microscope slide and cover slip.
2. Microscopy mounting medium.
3. Adhesive.
390
Nicholas Banahene and Benjamin M. Swarts
C for 10 min, allow to cool to room temperature,
and then aseptically add 100 mL of ADC supplement. Store at
4
C.
3. 1Â phosphate-buffered saline (1Â PBS): In a clean 2 L
graduated beaker, add 8 g NaCl, 0.2 g KCl, 1.44 g
Na 2 HPO 4 , and 0.24 g KH 2 PO 4 . Add 800 mL of ultrapure
water and stir to dissolve. Adjust the pH to 7.4 using HCl. Add
ultrapure water to a final volume of 1 L. Sterile-filter and store
at 4
C.
4. 0.5% bovine serum albumin in 1Â PBS (PBSB): Dissolve 0.5 g
of bovine serum albumin in 100 mL of 1Â PBS, sterile-filter,
and store at 4
C.
5. 4% formaldehyde in PBS: Protect from light and store at 4
C.
2.4 CopperCatalyzed Azide–
Alkyne Cycloaddition
(CuAAC) Reaction
1. 1 mM Az-488: Dissolve 1.0 mg of carboxyrhodamine
110 Azide (Click Chemistry Tools) in 1.7 mL of DMSO.
Vortex briefly to dissolve and store at À20
C (see Note 3).
2. 1 mM Alk-488: Dissolve 1.0 mg of carboxyrhodamine
110 Alkyne (Click Chemistry Tools) in 1.7 mL of DMSO.
Vortex briefly to dissolve and store at À20
C (see Note 3).
3. 60 mM sodium ascorbate: Dissolve11.9 mg of (+)-sodium Lascorbate (Millipore Sigma) in 1.0 mL of ultrapure water.
Vortex briefly to dissolve. Prepare fresh and use immediately
to avoid oxidation.
4. 6.4 mM TBTA: Dissolve 3.4 mg of tris(benzyltriazolylmethyl)amine (TBTA) ligand (Click Chemistry Tools) in 1.0 mL of tBuOH/DMSO (3:1 v/v). Vortex briefly to dissolve and store
at À20
C (see Note 4).
5. 50 mM Copper sulfate: Dissolve12.5 mg of copper(II) sulfate
pentahydrate (Millipore Sigma) in 1.0 mL of ultrapure water.
Vortex briefly to dissolve and store at 4
C.
2.5 Strain-Promoted
Azide–Alkyne
Cycloaddition (SPAAC)
and Tetrazine
Reactions
1. 1 mM Cy3 DBCO: Dissolve 1.0 mg of Cy3 DBCO (Click
Chemistry Tools) in 1.0 mL of DMSO. Vortex briefly to dissolve and store at À20
C (see Note 3).
2. 1 mM Cy3 tetrazine: Dissolve 1.0 mg of Cy3 tetrazine (Click
Chemistry Tools) in 1.1 mL of DMSO. Vortex briefly to dissolve and store at À20
C (see Note 3).
2.6 Fluorescence
Microscopy
1. Microscope slide and cover slip.
2. Microscopy mounting medium.
3. Adhesive.
390
Nicholas Banahene and Benjamin M. Swarts