UltraPourquoi et comment les trypanosomes construisent un Collier de la Poche FlagellaireAlliance française contre les maladies parasitaire

BONHIVERS, Mélanie

CASAS, Elina

LANDREIN, Nicolas

Oskar Bordeaux

HAL Id: hal-03880908https://hal.science/hal-03880908Preprint submitted on 20 Jan 2023HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.Ultra Expansion microscopy protocol with improvedsetup for upright and inverted microscopes. v1Elina Casas, Nicolas Landrein, Mélanie BonhiversTo cite this version:Elina Casas, Nicolas Landrein, Mélanie Bonhivers. Ultra Expansion microscopy protocol with im-proved setup for upright and inverted microscopes. v1. 2023. ￿hal-03880908￿JAN 26, 2022Ultra Expansion microscopy protocol with improvedsetup for upright and inverted microscopes.Elinacasas , Nicolas LANDREIN , Mélanie BonhiversMFP, CNRS UMR 5234, Université de Bordeaux, ProParaCyto group.Mélanie BonhiversDOI:dx.doi.org/10.17504/protocols.io.bvwqn7dwDocument Citation: Elinacasas, Nicolas LANDREIN, MélanieBonhivers 2022. UltraExpansion microscopyprotocol with improved setupfor upright and invertedmicroscopes.. protocols.iohttps://dx.doi.org/10.17504/protocols.io.bvwqn7dwLicense: This is an openaccess document distributedunder the terms ofthe Creative CommonsAttribution License,  whichpermits unrestricted use,distribution, and reproductionin any medium, provided theoriginal author and source arecreditedCreated: Jun 17, 2021Last Modified: Jan 26, 2022DOCUMENT integer ID:50864Keywords: Immuno-labelling,fluorescence microscopy,expansion microscopy, ultraexpansion microscopy, U-ExM, upright microscope,inverted microscope1 1 11ABSTRACTUltraprotocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 1Ultra Expansion microscopy This protocol is based on the following papers: CITATIONGambarotto D, Hamel V, Guichard P (2021). Ultrastructure expansion microscopy (U-ExM).. Methods in cell biology.LINKhttps://doi.org/10.1016/bs.mcb.2020.05.006CITATIONAmodeo 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.LINKhttps://doi.org/pii:jcs254300.10.1242/jcs.254300CITATIONIsch 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.LINKhttps://doi.org/10.1371/journal.ppat.1009329Reagents Company Reference CommentAbsolute ethanol Acrylamide 40% Euromedex EU0060-A store at 4°CAmmonium persulfate (APS) Euromedex EU0009 store as 10% in H2O at -20°CFormaldehyde 37% Sigma 252549HCl 1N solutionHOECHST (bisBenzimide H 33342 trihydrochloride)Sigma B2261 make 5 mg/mL in H2O. Store at -20°C as aliquotesprotocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 2milliQ Water 0.2 um filteredN,N’-Methylenbisacrylamide 2%Euromedex EU0560 store at 4°CPBS 0.2 um filteredSDS 20%Tris-BaseNaCl stock solution 5MSodium Acrylate 38% AK Scientific 97–99%, R624 store the powder at -20°CTEMED Euromedex 50406Tween-20 Sigma P-7949 store RTReagents Company Reference CommentEquipment Company Reference Comments12 mm round coverslips         VWR 6530021 CommentsMetal block pre-cooled -20°CShaker RTIncubator 37°C shaker or that can accomodate a shakerhomemade spatula with a rigid plastic cover1.5 ml tubes24 mm round coverslips  24-well plates NUNC6-well plates NUNCGlass slides Themoscientific J1800 AMN7Laboratory wipesPetri dishesPlumbing joints 20x27 N14Reagentsprotocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 3Thermoblock EppendorfVortexParafilmEquipment Company Reference Commentscompound volume Final concentrationFormaldehyde 37% 19 ul 0.7%Acrylamide 40% 25 ul 1%PBS 1x 956 ulPrepare just before useReagent B Final concentration CommentSodium acrylate 38% 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. Stock solution Stock solution Volume to prepare per sample Final concentrationSodium 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 1xEquipmentActivation solution (FA/AA)Sodium acrylate stock solution (SA)protocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 4Make 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.Stock solution Stock solution Volume to prepare per sample Final concentrationReagent Stock concentration Volume / Weight Final concentrationTRIS-Base - 0.6 g 50 mMHCl (fuming?) - pH to 9.0ddH2O - 10 mLSDS 20% (694 mM) 28.82 mL 200 mMNaCl 5 M 4 mL 200 mMmilliQ water qsp 100 mLDay 0.Preparation coverslips ( 24mm ) coated with poly-L-lysine for mounting- Place the coverslips on Parafilm and cover each of them with  400 µL   of poly-L-lysine 00:30:00  - 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 coverslipsIn 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  1 mL  of FA/AA solution- Fill up the empty wells with waterU-ExM Monomer solution (MS)Denaturation solution (DS)protocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 5- Incubate at 37 °C  100 rpm 04:00:00  3- Gel polymerizationa- Prepare On ice    - a lid of a 24-well plate covered with a pad and parafilm.    - A -20°C-pre-cooled metallic tube holder with             - 100 µL  of APS 10%             - 100 µL  of TEMED 10%            - a 90 µL  aliquot of MS solutionb- Prepare the MS + TEMED + APS solution and gel polymerization            - Add 5 µL  of TEMED 10% into the 90 ul MS aliquote, then add  5 µL  of APS 10%            - Vortex 2 sec and load on the parafilm 2 drops of 35 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 On ice  00:05:00  , then at 37 °C  without shaking 01:00:00  . During this step, prepare step 4.c-Gel detachment            - After the polymerization and in a 6-well plate, add 1 mL  of denaturation solution.              - Transfer the coverslip+polymerized gel (gel facing up ) to the 6-well plate            - Incubate  Room temperature  00:15:00  100 rpm or until the gel detaches from the coverslip.4- Denaturation- Set the Thermoblock at 95°C- Pre-heat at 95°C 1 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 01:30:00  .5- First expansion- Load each gel in a petri dish with 40 mL  milliQ water- Incubate 00:30:00  Room temperature  100 rpm  -Repeat twice  01:00:00  Room temperature  100 rpm  ( -OR  Change the water bath at least once and incubate Overnight 4 °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 00:10:00  Room temperature 100 rpm  - Repeat twice 00:20:00  Room temperature  100 rpm  a- In a 24-well plate, per sample, add 1 mL  of PBS, 2% BSA, 0.2% Tween-20.protocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 6 - 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 00:10:00 or more  37 °C  100 rpm  - Primary antibody(ies) incubation            - Remove the PBS, BSA, Tween-20 solution and replace it  with 350 µL of primary antibody(ies) diluted in PBS, BSA, Tween-20            - Incubate 37 °C Overnight  100 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  350 µL  PBS, BSA, Tween-20 37 °C  - Remove the PBS, BSA, Tween-20 solution and replace it with 350 µL  of secondary antibody(ies) and  Hoechst ( 5μg/mL ) diluted in PBS, BSA, Tween-20- Incubate 37 °C 03:00:00 100 rpm   , the lid sealed closed to avoid evaporation, in the dark.- Wash 3 times 10 min in 1 mL  PBS, BSA, Tween-20 37 °C  00:30:00  100 rpm  2- Second expansion- Transfer the gel into a 6-well plate and wash with 5 mL  water 00:30:00  OR ( - Change the water bath and incubate  Overnight   37 °C  100 rpm  ) - Change twice the water bath and incubate for 30 min 01:00:00  37 °C  100 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 imagingThis 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.protocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 7 How to mount your gel. protocols.io |https://dx.doi.org/10.17504/protocols.io.bvwqn7dw 8

Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes. v1

https://oskar-bordeaux.fr/bitstream/20.500.12278/199312/3/MFP_2023_Casas.pdf

Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes. v1

Abstract

Similar works

Full text

Available Versions

Oskar Bordeaux