Protocol Citation: miao.tian 2022. Conventional fixation method for Tetrahymena thermophila. protocols.io https://protocols.io/view/conventional-fixation-method-for-tetrahymena-therm-b45fqy3nVersion created by miao tian
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: In development
We are still developing and optimizing this protocol
Created: February 15, 2022
Last Modified: February 15, 2022
Protocol Integer ID: 58247
Keywords: fixing tetrahymena thermophila cell, tetrahymena thermophila cell, conventional fixation method for tetrahymena, thermophila, tetrahymena, conventional fixation method, cell
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Abstract
A rapid and robust method for fixing Tetrahymena thermophila cells
General description of the method
It works for the IF of cytoplasm, nucleoplasm, and chromatin-bound proteins, FISH staining of receptive sequences (e.g., telomeric repeats, TEs). Nuclear and cell morphology are nicely maintained.
It does not work for the IF of Cna1, γ-H2A.X, and tubulin.
Reagents
- 37% Formaldehyde (! Toxic, handel it in a fume hood)
- Triton X-100
- Sucrose
- Paraformaldehyde (!Toxic; Cat# A3813, 1000. Applichem)
Recipes for making solutions
17h
Preparing 4% formaldehyde/ 3.4% sucrose fixative 100 mL
Weight 4 g of paraformaldehyde, add 100 mL of water.
- Heat to dissolve paraformaldehyde in a fume hood. Namely, placing the flask on top of a 170 °C heat plate with stirring function, for around 00:25:00 , with stirring. Try not let it boil.
- Leave it on bench to cool for around 00:30:00 (do not make it completely cool, otherwise sugar dissolves very slow).
55m
Add 3.4 g of Sugar into the warm paraformaldehyde solution, stir/dissolve it.
- leave it at room temperature overnight and then store it in a fridge.
16h
10% Triton X-100 50 mL
- Add 5 mL of Triton X-100 into 45 mL of water. Microwave for 5s will let Triton X-100 dissolve rapidly.
Fixation of 5 mL of Tetrahymena cells
31m
(!) For growing cells: Wash cells once with 10 millimolar (mM) Tris-HCl (7.5 ) and resuspend cells with the same volume of 10 millimolar (mM) Tris-HCl (7.5 ). Starvation or conjugation cells need not to be washed for another time.
Shoot 250 µL of 10% Triton X-100 (first) and 500 µL of 37% formaldehyde into cells. Invert tube ~ 5 times and keep it at room temperature (25 °C ) for 00:30:00 ;
30m
Collect cells by centrifugation at 1500 rpm for 00:01:00 and pour supernatant (! contains formaldehyde, think about waste disposal rules)
1m
Resuspend cell pellet with ~ 500 µL of 4% formaldehyde/ 3.4% sucrose fixative, mixing cells by gentle pipetting is recommended.
Cells are ready to apply onto slides. After drying in a fume hood for 1hr, slides are ready for staining.