Aug 24, 2022

Public workspacePreparation and imaging of enriched Golgi from GolgiTAG-IP using Transmission Electron Microscopy

Proceedings of the National Academy of Sciences of the United States of America
DOI
dx.doi.org/10.17504/protocols.io.x54v9y9nqg3e/v1
  • 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK;
  • 2Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Dario R Alessi
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DOI: dx.doi.org/10.17504/protocols.io.x54v9y9nqg3e/v1
External link: https://doi.org/10.1073/pnas.2219953120
Protocol Citation: Rotimi Fasimoye, Alan Prescott, Dario R Alessi 2022. Preparation and imaging of enriched Golgi from GolgiTAG-IP using Transmission Electron Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9y9nqg3e/v1
Manuscript citation:
Fasimoye R, Dong W, Nirujogi RS, Rawat ES, Iguchi M, Nyame K, Phung TK, Bagnoli E, Prescott AR, Alessi DR, Abu-Remaileh M, Golgi-IP, a tool for multimodal analysis of Golgi molecular content. Proceedings of the National Academy of Sciences of the United States of America 120(20). doi: 10.1073/pnas.2219953120
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 22, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69022
Keywords: Enriched Golgi imaging, GolgiTAG-IP, Transmission Electron Microscopy, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000463
UK Medical Research Council
Grant ID: MC_UU_00018/1
Abstract
Transmission electron microscopy (TEM) is a tool used to image, in good resolution, the structure of organelles within the cell. Available protocols are designed to image structures within fixed intact cells. Here, we described a protocol where Golgi, isolated from cells by GolgiTAG immunoprecipitation (IP) (as described in ddx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1), can be prepared and imaged using TEM. This protocol can also be used to image any organelles isolated using various organelle-IP protocols that are available.
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Materials
Materials:

  • ReagentParaformaldehydeSigma AldrichCatalog #158127
  • ReagentGlutaraldehyde EM Grade 25%Sigma AldrichCatalog #G5882-50ML
  • ReagentSodium cacodylate trihydrateMerck Millipore SigmaCatalog #C0250
  • Osmium tetroxide (Agar. Catalog # R1023)
  • ReagentSodium ferrocyanide decahydrateSigma AldrichCatalog #13425
  • Tannic acid (BDH. Catalog # 30337)
  • Uranyl acetate (Agar. Catalog # R1260A)
  • ReagentDurcupan™ ACMSigma AldrichCatalog #44611
  • ReagentLead(II) citrate tribasic trihydrateSigma AldrichCatalog #15326
  • ReagentSodium citrate monobasicSigma AldrichCatalog #71497
  • ReagentSodium hydroxide (NaOH)Sigma AldrichCatalog #S5881
  • Potassium chloride (KCl) (VWR. Catalog# 267264)
  • ReagentPotassium phosphate monobasicSigma – AldrichCatalog #P5379
  • Calcium chloride (Calbiochem. Catalog# 208291)
  • Reagent(R)-( )-Propylene oxideSigma AldrichCatalog #540048
  • ReagentEthyl alcohol, 200 proof, anhydrous, ≥99.5%Sigma AldrichCatalog #459836

Buffer:

  • KPBS (Concentration136 millimolar (mM) KCl, Concentration10 millimolar (mM) KH2PO4. Adjust to pH 7.25 with KOH).
  • Concentration0.4 Molarity (M) sodium cacodylate buffer (Amount17.12 g sodium cacodylate dissolved in water. Adjust to pH7.4 with HCl).
  • Reynold lead citrate (Amount1.33 g lead citrate, Amount1.76 g sodium citrate; Amount8 mL 1N NaOH in Amount50 mL water).

Equipment:

  • ReagentPierce™ Anti-HA Magnetic BeadsThermo FisherCatalog #88837
  • ReagentDynaMag™- Spin MagnetThermo FisherCatalog #12320D
  • Microcentrifuge with thermostat (VWR Micro Star 17R. S/N 42209232)
  • Scalpel
  • P1000 pipette tips
  • Pasteur pipette
  • Oven (TAAB. I064)
  • Leica UCT ultramicrotome (Leica)
  • ReagentClear glass with solid top black phenolic 14B rubber lined capVWR international LtdCatalog #215-3571
  • JEOL 1200EX TEM using SIS III camera



Method
Method
2d 5h 4m
2d 5h 4m
Perform GolgiTAG-IP as previously described in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1
After last wash of beads with KPBS, fix the beads in Amount1 mL solution containing 4% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in Concentration0.1 Molarity (M) sodium cacodylate buffer dilute in water for Duration01:00:00 at TemperatureRoom temperature .

1h
Pipetting
Pellet beads with magnet DynaMagTM Spin Magnet (or equivalent) and remove the supernatant.
Wash beads pellet three times in Amount1 mL Concentration0.1 Molarity (M) sodium cacodylate buffer, pelleting the beads with magnet after each wash.

Wash
After third wash, spin down beads in tabletop centrifuge at Centrifigation1000 x g for Duration00:01:00 . Pellet should be tightly packed.

1m
Centrifigation
Use a needle to gently dislodge the pellet and use a Pasteur pipette to remove the pellet to a clean glass vial. (Note: All steps are carried out in a glass vial).
Pipetting
To ensure rapid dehydration and embedding, cut pellet with scalpel into approximately 1mm3 pieces, then post-fix in Amount1 mL 1% (w/v) Osmium tetroxide with 1.5% (w/v) sodium ferrocyanide in Concentration0.1 Molarity (M) sodium cacodylate buffer for Duration01:00:00 at TemperatureRoom temperature .

1h
Pipetting
Wash pellets three times in Concentration0.1 Molarity (M) sodium cacodylate buffer, by adding the buffer, allowing the pellets to settle and gently taking out supernatant with P1000 tip.
Wash
Wash pellets three times in Concentration0.1 Molarity (M) sodium cacodylate buffer for Duration00:01:00 (1/3).
1m
Centrifigation
Wash
Wash pellets three times in Concentration0.1 Molarity (M) sodium cacodylate buffer for Duration00:01:00 (2/3).
1m
Centrifigation
Wash
Wash pellets three times in Concentration0.1 Molarity (M) sodium cacodylate buffer for Duration00:01:00 (3/3).
1m
Centrifigation
Wash
After the third wash, wash with water three times (as in step 8), then resuspend in 1% w/v tannic acid and 1% w/v uranyl acetate diluted in water for Duration01:00:00 . Remove supernatant.


1h
Dehydrate pellet through alcohol series:
50% ethanol for Duration00:10:00
70% ethanol for Duration00:10:00
80% ethanol for Duration00:10:00
90% ethanol for Duration00:10:00
95% ethanol for Duration00:10:00
then twice in 100% ethanol for Duration00:10:00 (1/2)
100% ethanol for Duration00:10:00 (2/2)
Note
Use a P1000 pippete to gently remove ethanol after each series.


1h 10m
Pipetting
Further dehydrate in 100% propylene oxide twice for 10 min.

Further dehydrate in 100% propylene oxide twice for Duration00:10:00 (1/2).
10m
Further dehydrate in 100% propylene oxide twice for Duration00:10:00 (2/2).
10m
Resuspend pellet into 50% v/v propylene oxide and 50% v/v Durcupan resin DurationOvernight .

10m
Overnight
Open glass vial to allow propylene oxide to evaporate (This will take approximately Duration01:00:00 ).

1h
To embed pellet in resin, resuspend in 100% Durcupan resin.
Embed polymerise resin in an oven at Temperature60 °C for Duration48:00:00 .

2d
Cut sections of polymerised embedded resin with Leica UCT ultramicrotome.
Note
Sections should be 70-100nm thick

Stain sections with 3% aqueous uranyl acetate for Duration00:20:00 .

20m
Stain with Reynold lead citrate for Duration00:20:00 .

20m
Image stained sections on JEOL 1200EX TEM using SIS III camera.
Note
Note: Due to the cutting and many centrifugation steps, it is expected that the magnetic particles dissociate from the plastic bead core.)


Imaging