Feb 13, 2023

Public workspaceReynolds' Lead Citrate Stain

This protocol is a draft, published without a DOI.
  • 1University of Gothenburg
  • CCI_Gothenburg
Jens Berndtsson
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Protocol Citation: Jens Berndtsson 2023. Reynolds' Lead Citrate Stain. protocols.io https://protocols.io/view/reynolds-39-lead-citrate-stain-cmnyu5fw
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: January 12, 2023
Last Modified: February 13, 2023
Protocol Integer ID: 75192
Funders Acknowledgements:
National Microscopy Infrastructure
Grant ID: NMI (VR-RFI 2019-00217)
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Abstract
Reynolds Lead Citrate Stain for electron microscopy is a standard stain developed by Edward S. Reynolds and consists of a mixture of 80 mM Lead(II) nitrate and 120 mM Sodium citrate. Sodium hydroxide is added to make the stain basic to pH 12.

Preparation time:
40-60 min

Reference:
Edward S. Reynolds; THE USE OF LEAD CITRATE AT HIGH pH AS AN ELECTRON-OPAQUE STAIN IN ELECTRON MICROSCOPY .J Cell Biol1 April 1963; 17 (1): 208–212. doi:https://doi.org/10.1083/jcb.17.1.208
Guidelines
This protocols aim to show the exact method that the Centre for Cellular Imaging at the University of Gothenburg use to prepare a standard buffer.

We do not claim to have discovered, invented or otherwise hold any form of intellectual property of the buffer(s) described in this protocol.

Feel free to visit us: https://www.gu.se/en/core-facilities/centre-for-cellular-imaging
Materials
Chemicals
  • ReagentLead(II) nitrate (EMSURE®)Catalog #1073980100
  • ReagentTri-Sodium citrate dihydrate (EMSURE®)Catalog #1064480500
  • ReagentSodium hydroxide BioXtra ≥98% (acidimetric) pellets (anhydrous)Sigma AldrichCatalog #S8045-1KG
  • ReagentMilli-Q water

Consumables
  • 50 ml volumetric flask
  • Magnetic stirrer bead
  • 5 x 10 ml syringes
  • 10 ml pipette
  • Pasteur pipette
  • Parafilm
  • Weighing paper
  • Weighing spatula

Equipments/Machines
  • Scale
  • Magnetic stirrer
Protocol materials
ReagentLead(II) nitrate (EMSURE®)Merck MilliporeSigma (Sigma-Aldrich)Catalog #1073980100
ReagentTri-Sodium citrate dihydrate (EMSURE®)Merck MilliporeSigma (Sigma-Aldrich)Catalog #1064480500
ReagentSodium hydroxide BioXtra ≥98% (acidimetric) pellets (anhydrous)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S8045-1KG
ReagentMilli-Q water
ReagentSodium hydroxide BioXtra ≥98% (acidimetric) pellets (anhydrous)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S8045-1KG
ReagentLead(II) nitrate (EMSURE®)Merck MilliporeSigma (Sigma-Aldrich)Catalog #1073980100
ReagentTri-Sodium citrate dihydrate (EMSURE®)Merck MilliporeSigma (Sigma-Aldrich)Catalog #1064480500
ReagentTri-Sodium citrate dihydrate (EMSURE®)Merck MilliporeSigma (Sigma-Aldrich)Catalog #1064480500
ReagentSodium hydroxide BioXtra ≥98% (acidimetric) pellets (anhydrous)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S8045-1KG
ReagentMilli-Q water
ReagentMilli-Q water
ReagentLead(II) nitrate (EMSURE®)Merck MilliporeSigma (Sigma-Aldrich)Catalog #1073980100
ReagentMilli-Q water
Safety warnings
Use caution, work in a fume hood, wear gloves, lab coat, and goggles.

ReagentLead(II) nitrate (EMSURE®)Catalog #1073980100
H272 May intensify fire; oxidizer.
H302 + H332 Harmful if swallowed or if inhaled.
H317 May cause an allergic skin reaction.
H318 Causes serious eye damage.
H360FD May damage fertility. May damage the unborn child.
H372 Causes damage to organs (Blood, Central nervous system, Immune system, Kidney) through prolonged or repeated exposure.
H410 Very toxic to aquatic life with long lasting effects.

ReagentTri-Sodium citrate dihydrate (EMSURE®)Catalog #1064480500
Not a hazardous substance or mixture according to Regulation (EC) No 1272/2008.

ReagentSodium hydroxide BioXtra ≥98% (acidimetric) pellets (anhydrous)Sigma AldrichCatalog #S8045-1KG
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
Before start
Book a fume hood and label the syringes for aliquots.
50 ml Lead citrate for Array tomography
50 ml Lead citrate for Array tomography
31m
31m
Weigh out and dissolve Lead (II) nitrate and tri-Sodium citrate dihydrate:
  • Weigh out Amount1.32 g of ReagentLead(II) nitrate (EMSURE®)Sigma AldrichCatalog #1073980100 in a 50 ml volumetric flask in which you have added a small magnetic stirrer.
  • Dissolve the Lead(II) nitrate by adding Amount30 mL of ReagentMilli-Q water and turning on the stirrer.
  • Weigh out Amount1.76 g of ReagentTri-Sodium citrate dihydrate (EMSURE®)Sigma AldrichCatalog #1064480500 in a small weighing boat. Add it to the dissolved Lead(II) nitrate.
  • Shake the mixture vigorously, or vortex it for Duration00:01:00 The solution will be milky.
  • Cover the volumetric flask with parafilm and let it stir for an additional Duration00:30:00

Prepare a 1 M sodium hydroxide (NaOH) solution:
  • Add Amount10 mL of ReagentMilli-Q water to a 15 ml falcon tube.
  • Weigh out Amount0.4 g of ReagentSodium hydroxide BioXtra ≥98% (acidimetric) pellets (anhydrous)Sigma AldrichCatalog #S8045-1KG on a small weighing paper, and add the NaOH to the water.
  • Mix the NaOH until it dissolves by inverting the falcon tube.
Note
If your lab have a stock solution of NaOH, this can be diluted to 1 M and used instead of preparing it fresh as described above.

Finalizing the Lead citrate solution:
  • Add Amount8 mL of the 1 M NaOH solution to the Lead(II) nitrate/Tri-Sodium citrate mixture, this will raise the final pH to 12±0.1.
  • Keep stirring until the solution is clear.
  • Add ReagentMilli-Q water until the volume is 50 ml.

Aliquote the mixture in:
5 x Amount10 mL - 10 ml syringes labelled: Lead citrate and date.

Storage:
Mix on the day of use for optimal staining. For longer storage, seal syringes with parafilm and store long term (4 weeks) at Temperature4 °C
Note
If using stored lead citrate, carfully inspect before usage for precipitates. Do not use if precipitates are formed. Always ad a 0.2 µm filter to the syringe before use.

Clean up:
Rinse the 50 ml volumetric flask and magnetic stirrer twice with water, pouring the wash of in a liquid lead waste. Then rinse thouroghly with water before before putting them in the dishwasher.

31m