Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes.

Elina Casas; Nicolas  LANDREIN; Mélanie  Bonhivers

Jan 26, 2022

Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes.

DOI

dx.doi.org/10.17504/protocols.io.bvwqn7dw

¹MFP, CNRS UMR 5234, Université de Bordeaux, ProParaCyto group.

Mélanie Bonhivers

DOI: dx.doi.org/10.17504/protocols.io.bvwqn7dw

Document Citation: Elinacasas, Nicolas LANDREIN, Mélanie Bonhivers 2022. Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes.. protocols.io https://dx.doi.org/10.17504/protocols.io.bvwqn7dw

License: This is an open access document 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

Created: June 17, 2021

Last Modified: January 26, 2022

Document Integer ID: 50864

Keywords: Immuno-labelling, fluorescence microscopy, expansion microscopy, ultra expansion microscopy, U-ExM, upright microscope, inverted microscope

Funders Acknowledgements:

ANR PRCI Structu-Ring

Grant ID: ANR-20-CE91-0003

ANR LabEx ParaFrap

Grant ID: ANR-11-LABX-0024

Abstract

Ultra

Ultra Expansion microscopy 

This protocol is based on the following papers: 

CITATION
Gambarotto D, Hamel V, Guichard P (2021). Ultrastructure expansion microscopy (U-ExM).. Methods in cell biology.LINK
https://doi.org/10.1016/bs.mcb.2020.05.006

CITATION
Amodeo S, Kalichava A, Fradera-Sola A, Bertiaux-Lequoy E, Guichard P, Butter F, Ochsenreiter T (2021). Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.. Journal of cell science.LINK
https://doi.org/pii:jcs254300.10.1242/jcs.254300

CITATION
Isch C, Majneri P, Landrein N, Pivovarova Y, Lesigang J, Lauruol F, Robinson DR, Dong G, Bonhivers M (2021). Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar.. PLoS pathogens.LINK
https://doi.org/10.1371/journal.ppat.1009329



ReagentsCompanyReferenceComment
Absolute ethanol 
Acrylamide 40%EuromedexEU0060-Astore at 4°C
Ammonium persulfate (APS)EuromedexEU0009store as 10% in H2O at -20°C
Formaldehyde 37%Sigma252549
HCl1N solution
HOECHST (bisBenzimide H 33342 trihydrochloride)SigmaB2261make 5 mg/mL in H2O. Store at -20°C as aliquotes
milliQ Water0.2 um filtered
N,N’-Methylenbisacrylamide 2%EuromedexEU0560store at 4°C
PBS0.2 um filtered
SDS 20%
Tris-Base
NaClstock solution 5M
Sodium Acrylate 38%AK Scientific97–99%, R624store the powder at -20°C
TEMEDEuromedex50406
Tween-20SigmaP-7949store RT
Reagents



EquipmentCompanyReferenceComments
12 mm round coverslips         VWR6530021Comments
Metal blockpre-cooled -20°C
ShakerRT
Incubator 37°Cshaker or that can accomodate a shaker
homemade spatulawith a rigid plastic cover
1.5 ml tubes
24 mm round coverslips  
24-well platesNUNC
6-well platesNUNC
Glass slides ThemoscientificJ1800 AMN7
Laboratory wipes
Petri dishes
Plumbing joints20x27 N14
ThermoblockEppendorf
Vortex
Parafilm
Equipment



compoundvolumeFinal concentration
Formaldehyde 37%19 ul0.7%
Acrylamide 40%25 ul1%
PBS 1x956 ul
Prepare just before use
Activation solution (FA/AA)



ReagentBFinal concentrationComment
Sodium acrylate38% w/vDissolve little by little in milliQH2O and on ice under the fume hood.  0.22 um filtered.This solution is critical. Use only solution that appears clear or very slightly yellow. 
Sodium acrylate stock solution (SA)



Stock solutionStock solutionVolume to prepare per sampleFinal concentration
Sodium Acrylate (SA)38% (W/W)500ul23% (W/V)
Acrylamide (AA)40%250ul10% (W/V)
Bis-acrylamide (BIS)2%50 ul0.1% (W/V)
PBS10x100 ul1x
Make 90 ul aliquotes and store at -20°C for up to 2-3 weeks Make 90 µL aliquots and store at -20°C for up to 2-3 weeks maximum. Note: the solution does not freeze.
U-ExM Monomer solution (MS)


ReagentStock concentrationVolume / WeightFinal concentration
TRIS-Base-0.6 g50 mM
HCl (fuming?)-pH to 9.0
ddH2O-10 mL
SDS20% (694 mM)28.82 mL200 mM
NaCl5 M4 mL200 mM
milliQ waterqsp 100 mL
Denaturation solution (DS)

Day 0.

Preparation coverslips ( 24mm ) coated with poly-L-lysine for mounting
- Place the coverslips on Parafilm and cover each of them with  Amount400 µL    of poly-L-lysine Duration00:30:00   
- recover and store the poly-L-lysine solution
- wash the slides 2 times in a water bath
- Dry the coverslips up on adsorbing paper. 

- Pre-cool overnight at -20°C a metallic tube holder

- If required, prepare your cells on 24 mm coverslips to grow.

Day 1- Sample preparatio, first expansion and primary antibody incubation 

1- Prepare cells on 12 mm coverslips
In 24-well plates, prepare coverslips with your favourite cells. The cells density should not be too high to avoid non-isotropic expansion (non-confluent cells).

Wash the cells with PBS.

2- Activation
- Place the coverslips in 24-well plate, cells up, and load  Amount1 mL   of FA/AA solution
- Fill up the empty wells with water
- Incubate at Temperature37 °C   Shaker100 rpm Duration04:00:00   

3- Gel polymerization
a- Prepare TemperatureOn ice  
    - a lid of a 24-well plate covered with a pad and parafilm.
    - A -20°C-pre-cooled metallic tube holder with 
            - Amount100 µL   of APS 10% 
            - Amount100 µL   of TEMED 10%
            - a Amount90 µL   aliquot of MS solution
b- Prepare the MS + TEMED + APS solution and gel polymerization
            - Add Amount5 µL   of TEMED 10% into the 90 ul MS aliquote, then add  Amount5 µL   of APS 10%
            - Vortex 2 sec and load on the parafilm 2 drops of Amount35 µL not more . (Do 2 drops at once only as the polymerization is very rapid).
            - Immediately, adsorb the excess FA/AA solution on a tissue and transfer the coverslips on the drop, cells side facing down.
            - Incubate TemperatureOn ice   Duration00:05:00   , then at Temperature37 °C   without shakingDuration01:00:00   . During this step, prepare step 4.
c-Gel detachment
            - After the polymerization and in a 6-well plate, add Amount1 mL   of denaturation solution.  
            - Transfer the coverslip+polymerized gel (gel facing up ) to the 6-well plate
            - Incubate  TemperatureRoom temperature   Duration00:15:00   Shaker100 rpm or until the gel detaches from the coverslip.

4- Denaturation
- Set the Thermoblock at 95°C
- Pre-heat at 95°C Amount1 mL   of DS per sample in an eppendorf tube. 
- Collect the detached gel and load it into the DS. 
- Fill up the tube with heated DS and incubate at 95°C Duration01:30:00   .

5- First expansion
- Load each gel in a petri dish with Amount40 mL   milliQ water
- Incubate Duration00:30:00   TemperatureRoom temperature   Shaker100 rpm  
-Repeat twice  Duration01:00:00   TemperatureRoom temperature   Shaker100 rpm  
( -OR  Change the water bath at least once and incubate DurationOvernight  Temperature4 °C  )
- Measure the diameter of the gel (expect of a >4x expansion compared to the 12mm diameter of the coverslip)


 6- Primary antibody incubation
- Incubate the gel in 40 mL PBS Duration00:10:00   TemperatureRoom temperature  Shaker100 rpm  
- Repeat twiceDuration00:20:00   TemperatureRoom temperature   Shaker100 rpm  a- In a 24-well plate, per sample, add Amount1 mL   of PBS, 2% BSA, 0.2% Tween-20.
 - Fill up the empty wells with water
- Gel cutting 
            - Use a 1 mL Tips to cut a circular piece of the gel ( usually 4 pieces can easily be cut from one gel).
            - Place each piece of gel into the 24-well plate 
            - Incubate in PBS, BSA, Tween-20 Duration00:10:00 or more  Temperature37 °C   Shaker100 rpm  
- Primary antibody(ies) incubation
            - Remove the PBS, BSA, Tween-20 solution and replace it  with Amount350 µL  of primary antibody(ies) diluted in PBS, BSA, Tween-20
            - Incubate Temperature37 °C  DurationOvernight   Shaker100 rpm  , the lid sealed closed to avoid evaporation.
         

Day-2 Secondary incubation, second expansion and gel mount

 1- Secondary antibody incubation
- Wash 3 times in  Amount350 µL   PBS, BSA, Tween-20 Temperature37 °C   
- Remove the PBS, BSA, Tween-20 solution and replace it with Amount350 µL   of secondary antibody(ies) and  Hoechst ( 5μg/mL ) diluted in PBS, BSA, Tween-20
- Incubate Temperature37 °C  Duration03:00:00  Shaker100 rpm   , the lid sealed closed to avoid evaporation, in the dark.
- Wash 3 times 10 min in Amount1 mL   PBS, BSA, Tween-20 Temperature37 °C   Duration00:30:00   Shaker100 rpm  

2- Second expansion
- Transfer the gel into a 6-well plate and wash with Amount5 mL   water Duration00:30:00   
OR ( - Change the water bath and incubate  DurationOvernight    Temperature37 °C   Shaker100 rpm  ) 
- Change twice the water bath and incubate for 30 min Duration01:00:00   Temperature37 °C   Shaker100 rpm  
- Measure the diameter of the gel, it should be consistent with the first expansion

( You can store the gels water in water at 4°C)

3- Mounting and imaging

This setup allows the imaging on upright and inverted microscopes. Also, the gels do not dehydrate during long microscopy sessions and can be further stored t 4°C.
How to mount your gel. 

Citations

Isch C, Majneri P, Landrein N, Pivovarova Y, Lesigang J, Lauruol F, Robinson DR, Dong G, Bonhivers M. Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar.

https://doi.org/10.1371/journal.ppat.1009329

Amodeo S, Kalichava A, Fradera-Sola A, Bertiaux-Lequoy E, Guichard P, Butter F, Ochsenreiter T. Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.

https://doi.org/pii:jcs254300.10.1242/jcs.254300

Gambarotto D, Hamel V, Guichard P. Ultrastructure expansion microscopy (U-ExM).

https://doi.org/10.1016/bs.mcb.2020.05.006

Reagents	Company	Reference	Comment
Absolute ethanol
Acrylamide 40%	Euromedex	EU0060-A	store at 4°C
Ammonium persulfate (APS)	Euromedex	EU0009	store as 10% in H2O at -20°C
Formaldehyde 37%	Sigma	252549
HCl			1N solution
HOECHST (bisBenzimide H 33342 trihydrochloride)	Sigma	B2261	make 5 mg/mL in H2O. Store at -20°C as aliquotes
milliQ Water			0.2 um filtered
N,N’-Methylenbisacrylamide 2%	Euromedex	EU0560	store at 4°C
PBS			0.2 um filtered
SDS 20%
Tris-Base
NaCl			stock solution 5M
Sodium Acrylate 38%	AK Scientific	97–99%, R624	store the powder at -20°C
TEMED	Euromedex	50406
Tween-20	Sigma	P-7949	store RT

Equipment	Company	Reference	Comments
12 mm round coverslips	VWR	6530021	Comments
Metal block			pre-cooled -20°C
Shaker			RT
Incubator 37°C			shaker or that can accomodate a shaker
homemade spatula			with a rigid plastic cover
1.5 ml tubes
24 mm round coverslips
24-well plates	NUNC
6-well plates	NUNC
Glass slides	Themoscientific	J1800 AMN7
Laboratory wipes
Petri dishes
Plumbing joints		20x27 N14
Thermoblock	Eppendorf
Vortex
Parafilm

compound	volume	Final concentration
Formaldehyde 37%	19 ul	0.7%
Acrylamide 40%	25 ul	1%
PBS 1x	956 ul
Prepare just before use

Reagent	B	Final concentration	Comment
Sodium acrylate		38% w/v	Dissolve little by little in milliQH2O and on ice under the fume hood. 0.22 um filtered.This solution is critical. Use only solution that appears clear or very slightly yellow.

Stock solution	Stock solution	Volume to prepare per sample	Final concentration
Sodium Acrylate (SA)	38% (W/W)	500ul	23% (W/V)
Acrylamide (AA)	40%	250ul	10% (W/V)
Bis-acrylamide (BIS)	2%	50 ul	0.1% (W/V)
PBS	10x	100 ul	1x
Make 90 ul aliquotes and store at -20°C for up to 2-3 weeks Make 90 µL aliquots and store at -20°C for up to 2-3 weeks maximum. Note: the solution does not freeze.

Reagent	Stock concentration	Volume / Weight	Final concentration
TRIS-Base	-	0.6 g	50 mM
HCl (fuming?)	-	pH to 9.0
ddH2O	-	10 mL
SDS	20% (694 mM)	28.82 mL	200 mM
NaCl	5 M	4 mL	200 mM
milliQ water		qsp 100 mL

Public workspaceUltra Expansion microscopy protocol with improved setup for upright and inverted microscopes.

Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes.