Plant actin visualization using expansion microscopy with Agrisera anti-actin monoclonal antibodies

Sample: Maize protoplasts fixed in 4% paraformaldehyde in 1X PBS pH 7.4 for 1 hour and washed 3 times in 1XPBS buffer

Primary antibodies: Agrisera AS21 4615 actin monoclonal; clone 14H4G8;

Secondary antibodies: Agrisera Donkey anti-Mouse IgG (H&L), DyLight® 488 conjugated AS10 1201

Other reagents: 30% Bovine Serum Albumin solution, Sigma Aldrich Cat # A8577-50ML; Phosphate Buffered Saline 10X, Electron Microscopy Sciences Cat # 19342-10; Imaging spacer, Millipore Sigma Grace Bio-Labs SecureSeal™ imaging spacer Cat # GBL654008

Protocol:

1. Block isolated protoplasts in a blocking buffer (2% BSA in 1x PBS) for 1 h at room temperature.
2. Dilute the primary antibody to 1: 500 in a blocking buffer, mix well, and spin down at 150 x g for 3 min:
3. Spin down protoplasts in a blocking buffer at 150 g for 1 -3 min and remove as much supernatant as possible using a pipette tip with a cut end (wide opening).
4. Resuspend protoplasts in diluted antibody and incubate for 1 h at room temperature.
5. Spin down protoplasts at 150 g for 1 min and remove as much supernatant as possible using a pipette tip with a cut end.
6. Wash protoplasts in a blocking buffer 3 times for 5 minutes each. Spin and resuspend as described in step 5 between each washing step.
7. Dilute the secondary antibody AS10 1201 to 1: 400, mix well, and spin down at 150 x g for 3 min:
8. Incubate protoplasts in diluted antibody for 1 h at room temperature.
9. Repeat the washing steps as described in 5-6.
10. Resuspend a protoplast pellet in a small volume of PBS 1X, pH 7.4.
11. Prepare microscopy slide: attach imaging spacer on top of the slide, peal off top adhesive membrane, add protoplast suspension on to the slide inside the spacer opening, cover with 22x22 mm cover glass No 1.5.
12. Imaging: Zeiss Elyra 7 SIM

Courtesy of Dr. Anastasiya Klebanovych, Dr. Kirk Czymmek, Dr. Kevin Cox, Dr. Blake Meyers, at the Donald Danforth Plant Science Center, USA