Oct 24, 2024

Public workspaceeasyIRF preparation of cell monolayers for in resin fluorescence using progressive lowering of temperature

DOI
dx.doi.org/10.17504/protocols.io.3byl498x8go5/v1
  • Dumisile Lumkwana1,
  • Marie-Charlotte Domart1,
  • Lucy Collinson1
  • 1The Francis Crick Institute
Dumisile Lumkwana
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DOI: dx.doi.org/10.17504/protocols.io.3byl498x8go5/v1
Protocol Citation: Dumisile Lumkwana, Marie-Charlotte Domart, Lucy Collinson 2024. easyIRF preparation of cell monolayers for in resin fluorescence using progressive lowering of temperature. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl498x8go5/v1
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: August 07, 2024
Last Modified: October 24, 2024
Protocol Integer ID: 106865
Keywords: easyIRF, Cell, Monolayer, Chemical fixation, Formaldehyde, Acetone, LR White, In Resin Fluorescence, Progressive Lowering of Temperature, IRF, PLT, Electron Microscopy, EM, Correlative light and electron microscopy, CLEM
Funders Acknowledgement:
Chan Zuckerberg Initiative
Grant ID: 2021-234618
Francis Crick Institute (Wellcome, MRC, CRUK)
Grant ID: CC1076
Disclaimer
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind.
Disclaimer statement source dx.doi.org/10.17504/protocols.io.bh9nj95e
Abstract
This easyIRF protocol details the preparation of cell monolayers for in resin fluorescence using progressive lowering of temperature.
Guidelines
Health & Safety requirements:
Ensure you are familiar with all relevant local Health and Safety documents and procedures (Material Safety Data Sheets, Procedural Risk Assessments, Standard Operating Procedures etc.) before starting.

Procedural Risk Assessments: Handling of formaldehyde fixative, benzoin methyl ether, LR White resin, liquid nitrogen.

Abbreviations:
  • IRF: In Resin Fluorescence
  • PLT: Progressive Lowering of Temperature
  • RT: Room Temperature
  • AFS: Automated Freeze Substitution
Materials
Materials

  1. Incubator set to 37ºC, 5% CO2
  2. Automated Freeze Substitution unit
  3. Fridge at 4ºC
  4. Vortex (for mixing solutions)
  5. pH meter
  6. Gilson P10ML pipette and tips
  7. Gilson P1000, 1 ml pipette and tips
  8. ReagentPlastic Transfer Pipette 3ml SLSCatalog #PIP4206
  9. Reagent15 mL centrifuge tubes, polypropyleneMerck MilliporeSigma (Sigma-Aldrich)Catalog #CLS430791
  10. Reagent50 mL PP centrifuge tubes, Polypropylene tubeMerck MilliporeSigma (Sigma-Aldrich)Catalog #CLS430921
  11. Magnetic stirrer plate
  12. Magnetic stirring bars (for stirring buffers)
  13. Measuring cylinders (for HEPES buffer prep)
  14. 500 ml and 100 ml glass beakers (for HEPES buffer prep)
  15. ReagentNalgene™ Rapid-Flow™ Sterile Disposable Filter Units with Nylon Membrane, 500mL, 0.2μm poreThermo FisherCatalog #151-4020
  16. 20 ml syringes (for prep of small volumes of HEPES buffer)
  17. 22 µm filter (for prep of small volumes of HEPES buffer)
  18. 15 ml glass scintillation vials with caps (for making up acetone & LR White)
  19. ReagentParafilmagar scientificCatalog #AGG3398
  20. ReagentDumont Tweezers 7agar scientificCatalog #AGT5039
  21. ReagentAluminium Dishesagar scientificCatalog # AGG3912
  22. Optional: ReagentDISPOSABLE WEIGHING DISH-144TAABCatalog #W083
  23. Optional: Reagent 35 mm TC-treated Culture DishCorningCatalog #430165
  24. ReagentAclar Filmagar scientificCatalog #AGL4458

Chemicals

  1. ddH2O; double distilled water
  2. Liquid Nitrogen
  3. ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
  4. Sodium hydroxide pellets
  5. Reagent16% ParaformaldehydeElectron Microscopy SciencesCatalog #15710
  6. Acetone (>=99.8%) Fisher Scientific 10252232
  7. Benzoin methyl ether
  8. ReagentLR White Resinagar scientificCatalog #AGR1280

Reagents

  1. HEPES buffer 0.8 M pH 7.4
  2. HEPES buffer 0.2 M pH 7.4
  3. Double strength fix: 8% formaldehyde in 0.4 M HEPES buffer – made fresh
  4. Single strength fix: 4% formaldehyde in 0.2 M HEPES buffer – made fresh
  5. Acetone dehydration series: 50%, 70%, 90%, 100% (dry) acetone
  6. LR White infiltration series: 25%, 50%, 75%, 100% – made fresh


HEPES buffer

Stock solution: 0.8 M HEPES buffer (pH 7.4)
  1. Dissolve 9.54 g of HEPES powder (>99.5%; Sigma-Aldrich H3375) in 40 ml of ddH2O
  2. Adjust the pH to 7.4 with sodium hydroxide (NaOH)
  3. Top up to 50 ml with ddH2O
  4. Filter sterilise and store at 4ºC

Working solution: 0.2 M HEPES buffer (pH 7.4)
  1. Add 12.5 ml of 0.8 M HEPES to 37.5 ml of ddH2O
  2. Store at 4ºC


Double strength fix, make fresh Safety critical

Working solution: 8% EM grade formaldehyde in ~0.4 M HEPES buffer pH 7.4
  1. Add 16% EM grade formaldehyde to 0.8 M HEPES stock solution, 1:1 (v:v).




Single strength fix, make fresh Safety critical

Working solution: 4% EM grade formaldehyde in ~0.2 M HEPES buffer pH 7.4
  1. Add 16% EM grade formaldehyde to 0.8 M HEPES stock solution and top up with ddH2O
according to the final volume required (Table 1).



100% dry acetone

  1. Transfer molecular sieves to a 500 ml glass bottle, add 99-100% acetone until it’s brim full, mix, and leave to settle.



Photo-activated LR White Resin, make fresh Safety critical

  1. Add 15 ml of catalysed LR White into a 15 ml glass vial
  2. Add 0.5-1% benzoin methyl ether (0.75 % = 0.1125g) to LR White and add a cap to the vial
  3. Cover with foil and place on the rotator
  4. Once dissolved, place in the fridge until use



















Safety warnings
Safety critical: All procedures designated safety critical must be performed in a fume hood (where appropriate) and the following personal protective equipment (PPE) must be worn - lab coat, gloves, and eye protection.
Procedure: Processing samples
Procedure: Processing samples

Note
  • This protocol was developed for cell monolayers grown on glass coverslips in 35 mm dishes. Appropriate solution volumes are indicated.
  • The protocol can be adapted for use on tissues by extending dehydration and infiltration times.
  • For dehydration and resin infiltration steps in metal dishes, fill the metal dish at least half full so that the cells will not dry out, which would destroy the ultrastructure.

Day 1
Day 1
1d 0h 15m
1d 0h 15m
Prepare fresh fixative solutions before starting with sample preparation Safety critical

  • Pre-warm double strength fix to Temperature37 °C (or the temperature at which the cells were grown)

Add an equal volume of pre-warmed double strength fix to the cells in medium and fix for Duration00:15:00 at TemperatureRoom temperature Safety critical

15m
Replace double strength fix with Amount2 mL of single strength fix and leave for Duration24:00:00 at Temperature4 °C Safety critical

1d
Day 2
Day 2
15m
15m
Prepare the Automated Freeze Substitution (AFS) unit Safety critical

  • Set the AFS unit to Temperature4 °C and fill with liquid nitrogen
Prepare solutions fresh before resuming the protocol Safety critical

Prepare graded acetone series and pre-cool in the fridge at Temperature4 °C

Prepare graded photo-activated LR white series and pre-cool in the fridge at Temperature4 °C

Remove fixative solution from the 35 mm dishes containing the coverslips and replace with Amount2 mL of Concentration0.2 Molarity (M) HEPES, TemperatureOn ice Safety critical

Wash the coverslips in:
Wash
Amount2 mL of Concentration0.2 Molarity (M) HEPES, for Duration00:05:00 , TemperatureOn ice (1/3)

5m
Wash
Amount2 mL of Concentration0.2 Molarity (M) HEPES, for Duration00:05:00 , TemperatureOn ice (2/3)

5m
Wash
Amount2 mL of Concentration0.2 Molarity (M) HEPES, for Duration00:05:00 , TemperatureOn ice (3/3)

5m
Wash

Note
Dehydration and infiltration notes:
  • Transfer pre-cooled 70% and 90% acetone to the AFS chamber. Also add an empty 15 ml glass bottle for waste.
  • After each solution change, remove the waste, and transfer the next solution from the fridge to the AFS unit so that it can pre-cool to the appropriate temperature.
  • Use pre-cooled metal forceps (orange insulated tweezers) to manipulate samples within the AFS unit.
  • Ensure there is enough acetone in the dishes - do not allow the cells to dry out as this will destroy the ultrastructure.

Transfer coverslips to metal dishes filled with pre-cooled 50% acetone TemperatureOn ice , and transfer to the AFS unit

Reduce the temperature in the AFS over Duration00:20:00 from Temperature4 °C to Temperature-20 °C

20m
Dehydrate cells stepwise at Temperature-20 °C in:

Pre-cooled 70% acetone for Duration00:10:00

10m
Pre-cooled 90% acetone for Duration00:10:00

10m
Pre-cooled anhydrous acetone for Duration00:10:00

10m
Pre-cooled anhydrous acetone for Duration00:10:00

10m
Infiltrate cells with a graded photo-activated LR White series at Temperature-20 °C in the dark Safety critical

Note
We suggest to cover the AFS lid with a box during infiltration to exclude light.

Pre-cooled 25% LR White for Duration00:10:00 .

10m
Pre-cooled 50% LR White for Duration00:10:00 .

10m
Pre-cooled 75% LR White for Duration00:10:00 .

10m
Pre-cooled 100% LR White DurationOvernight .

Day 3
Day 3
2d 18h 40m
2d 18h 40m
Make fresh photo-activated 100% LR White, pre-cool at Temperature4 °C for Duration00:20:00 and then transfer to the AFS unit for at least Duration00:20:00 before use to cool further to Temperature-20 °C Safety critical

40m
Infiltrate cells with the pre-cooled photo-activated 100% LR White for Duration02:00:00 at Temperature-20 °C in the dark

2h
Polymerise resin:

Place an Aclar disc (cut to size from an Aclar sheet) over the dish to exclude oxygen during polymerisation

If needed, top up the liquid nitrogen in the AFS, being careful not to decrease temperature below Temperature-20 °C Safety critical

Attach the UV lid to the AFS unit and turn on the UV power

Polymerise for Duration48:00:00 at Temperature-20 °C with the UV light on

2d
Warm samples to Temperature4 °C over Duration16:00:00 with the UV light turned off

16h
Store resin blocks in the fridge at Temperature4 °C