High pressure freezing and freeze substitution of cells grown on Aclar discs (Burger lab)

From LipidomicsWiki

Improved protocol based on the original published in;
Jimenez, N., Humbel, B.M., van Donselaar, E., Verkleij, A.J. & Burger, K.N.J. (2006) Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers. Journal of Microscopy 221, 216–223.

Preparation of culture substrates
Films of 51µm (2mil) thick Aclar®33C were purchased from Electron Microscopy Sciences (#50426-10; EMS, Fort Washington, PA).
Preparation of cell culture substrates, wear gloves:

• Cut out a 2 by 5 cm piece of Aclar film (with ordinary scissors), clean both sides using a lens cleaning tissue paper moistened with acetone and place between two lens cleaning tissue papers
• Punch out 1.5 mm discs using a biopsy punch (Acuderm inc. USA) and pull the discs out from the punch using a forceps, take care that the forceps only touches the edges of the discs.
• Remove the protective lens cleaning paper with a second forceps and store the Aclar disks in a glass container.
  

Seeding cells on Aclar disks

• Aclar disks are placed in 35mm culture dishes (8 disks per dish) containing 400µl supplement-free culture medium and attached to the dish using the capillary force.
  The location of the disks is marked on the underside of the culture dish.
  
• Culture dishes containing the Aclar disks are incubated for 2 h at 37°C to wash the disks and improve their hydrophilic character.
  
• Aspirate the supplement-free culture medium used in the pre-incubation of the Aclar discs and seed the dishes with cells harvested by trypsinization from flasks containing 80% confluent cultures in the desired dilution.
  
• Let the dishes rest for at least 15 minutes before moving them to the incubator.
  
• Avoid moving the dishes during the first few hours of incubation.
  
• Make sure the Aclar discs are not overgrown, this causes problems during embedding

High-pressure freezing
For HPF, Aclar discs are sandwiched between Ø 3×0.5 mm aluminium platelets
(M. Wohlwend, Sennwald, Switzerland) And high pressure frozen using a Leica high-pressure freezer (Leica EM HPF; Leica Microsystems, Vienna, Austria; now M. Wohlwend, Sennwald, Switzerland)

• Mount a platelet with two cavities, Ø 2×0.2 mm and Ø 2×0.1 mm, in the HPF specimen holder with the shallow cavity upwards.
  
• Using a forceps, remove an Aclar disk form the culture dish and place it in the shallow cavity with the cells facing up.
  
• Place the second platelet with a single cavity (Ø 2×0.3 mm) on top of the first platelet, with its flat surface facing down.
  
• Close the specimen holder and freeze the sample at a pressure of 2045 bar to a temperature of -160°C.
  
• After freezing, the intact ‘sandwich’ can be stored in liquid nitrogen.
  

Freeze substitution and embedding
Freeze substitution of the samples is carried out in microtubes (1.5 mL) containing flow-trough baskets (Leica Microsystems, Vienna, Austria) using a Leica AFS (Leica Microsystems, Vienna, Austria).

The program for freeze substitution is as follows; 46h at -90°C, transition to -60°C at 2°C/h (15h), 8h at -60°C, transition to -30 at 2°C/h (15h), 8h at -30°C.

The addition of 1 to 3% H2O to the freeze substitution medium enhances the retention of structure (Buser, C., Walther, P. (2008)) and increases contrast and membrane visibility.

• Prepare FS medium containing; 2% osmium tetroxide (EMS), 0.5% anhydrous glutaraldehyde (EMS) and 1 or 3% H2O in anhydrous acetone (#1.00299.0500; Merck, Darmstadt, Germany)
  
• Dispense 1 mL FS medium per microtube and immediately freeze in liquid nitrogen
  
• Transfer intact high-pressure frozen sandwiches to the microtubes under liquid nitrogen and place the microtubes in the pre-cooled AFS.
  
• After freeze substitution wash samples 2 times on ice and in the dark for 30 minutes with anhydrous acetone by transferring the baskets to a new microtube filled with anhydrous acetone.
  
• For Epon (#45359; Fluka) infiltration, incubate baskets at 4°C in microtubes with acetone : Epon mixtures 2 : 1 for 5h; 1 : 1 overnight; 1 : 2 for 24h; 1 : 3 for 4h
  
• Transfer baskets to pure Epon resin and incubate overnight at room temperature.
  
• Remove sandwiches from the baskets and disassemble in a dish with Epon resin.
  
• Transfer Aclar discs with cells facing upwards to a new dish with a 2-mm-thick layer of fresh Epon resin for flat embedding
  
• Immerse a 1-cm-long plastic cylinder, prepared from a Beem embedding capsule (#69911; EMS; bottom and lid cut off using a razor blade), in the resin on top of every Aclar disc. Let the resin polymerize overnight at 60°C.

• After polymerization fill up the cylinders with fresh Epon and leave at 60°C for another 24 h.
  
• Separate the polymerized blocks, with Aclar discs on their surface, from the dish. Trim to expose the Aclar disc separate the Aclar disc from the Epon block which is now ready for sectioning.