Sep 02, 2020
Agarose pads for microscopy
- 1Ronin Institute
Protocol Citation: Joao Vitor Molino 2020. Agarose pads for microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.bkn8kvhw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 02, 2020
Last Modified: September 02, 2020
Protocol Integer ID: 41408
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Abstract
This protocols describe the steps required for the preparation of agarose pads for live cell fluorescent microscopy.
Guidelines
All steps described in this protocol are intended to be conducted in a research laboratory. Follow aseptic procedures.
Materials
MATERIALS
Frame-Seal™ in situ PCR and Hybridization Slide Chambers 15 x 15 mm 65 µlCatalog #SLF0601
Safety warnings
Follow the biosafety guidelines for the cell lines use during the experiment.
Before start
Separate all material needed for the protocol.
Read through the protocol.
Before start
Before start
10m
10m
Check all the necessary material:
- Glass slide
- Cover slip
- Agarose (Agar should work as well, but probably with less imaging quality)
- Media or buffer
- Chamber or well forming stick (ex: Frame-Seal™ in situ PCR and Hybridization Slide Chambers, 15 x 15 mm, 65 µl)
- Tube or flask for melting agarose
- 1 mL plastic tips
Melting agarose
Melting agarose
5m
5m
- Melt the agarose in the desired concentration (1.5 Mass / % volume were tested) using a microwave, with interval mixing. Using the desired liquid. (e.g. Media for keeping the cells alive)
- Keep the melted agarose in a plate heater at 70 °C .
Glass slide preparation
Glass slide preparation
10m
10m
- Cut a 1mL tips to increase the point diameter for easier liquid handling of the agarose.
- Separate a glass slide to use as cover during pad preparation
- Stick the chamber forming material to a glass slide, maintaining the plastic barrier in the exposed side (Alternativaly you can follow the instructions at dx.doi.org/10.17504/protocols.io.kfrctm6 for a option without the chamber sticks)
- Add a excess of melted agarose to the well, and cover it with another glass slide for shaping the agarose pad
- Remove the glass slide, by sliding it carefully to the side
Cutted plastic tip
Melted agarose added
Solidifying agarose
Glass slide removed after solidification
Sample addition
Sample addition
10m
10m
- Add the desired amount of sample to the agarose pad. (5-61 µL drops were tested | Be aware of cell concentration if the goal is to see individual cells)
- Wait the drops to dry 00:05:00 to 00:10:00 (Specially important for cells with motility)
- Carefully remove the plastic barrier exposing the adhesive face of the well forming material
- Add a cover slip, sealing the well. (Avoids dehydration of the agarose pad, important for long microscopy experiments)
Samples added to agarose pad
Glass slide ready for microscopy