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New Leica EM AFS2/EM FSP Flow-through inserts for 6mm sample carriers

This new insert is suitable for manual freeze substitution process in the Leica EM AFS2 and for automated freeze substitution performed in conjunction with the Leica EM FSP unit.

1: Flow-through inserts for 6mm sample carriers

It is fully compatible with the reagent baths currently in use (16707154) and it is specially designed to accommodate all 6 mm carriers used for sample freezing in the Leica EM HPM100:

  • 6mm sapphire discs (16770158) and
  • 6 mm planchette carriers both the aluminum sample carriers (16770126 and16770127) and
  • the copper-gold plated carriers (16770181 and 16770182).

Manual

Please note that in the case of manual filling, changing or discarding of fluids should be done only via the central inlet (marked with asterisk on the Fig.2). Overspill/contamination of the inner segment (arrow head) should be avoided as it will lead to poor polymerization of the resin.

Use fine forceps to carefully position the 6mm carriers (cells/sample facing up) in the individual baskets within the flow-through insert.

To avoid re-crystallization or drying of the samples make sure the insert is filled with 4 ml substitution medium, pre-cooled to the appropriate temperature, prior to loading the sample.

After polymerization is completed release the flow-through insert from the reagent bath. Make incisions with a razor blade (Fig.3) to enable the separation of the flow-through insert from the reagent bath.

Next, clean the bottom part of the flow-through insert from excess resin (Fig.4).

Remove the resin between the baskets (Fig.6).

Make an incision with a razor blade in the flow-through insert from both sides of the basket, as shown on figure 7 (to the right ). Do not cut completely through! The incision should be half the length of the basket.

With the help of forceps bend the resin block and release it from the basket (Fig.8).

Once the block is released from the mold, gently remove the fine layer of resin covering the sapphire disc (Fig.9).

Make sure there is no residual resin surrounding the rim of the sapphire and cool down the sapphire by dipping it shortly into LN2. Gently lift the sapphire with the tip of the razor blade to separate it from the resin block (Fig.10).

The sample is ready to be placed in a universal specimen holder (16701761) for further trimming and sectioning.

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2: Please note that in the case of manual filling, changing or discarding of fluids should be done only via the central inlet (asterisk). Overspill/contamination of the inner segment (arrow head) should be avoided as it will lead to poor polymerization of the resin.

3: After polymerization is completed release the flow-through insert from the reagent bath. Make incisions with a razor blade to enable the separation of the flow-through insert from the reagent bath.

4: Clean the bottom part of the flow-through insert from excess resin. A = Layer of resin, B= Bottom side oft he mold , C = razor blade

5: Bottom side without the resin.

6: Remove the excess resin between all baskets (D).

7: Make an incision with a razor blade. Incision should be half the length of the basket. C = Razor blade.

8: Bend the resin block and release it from the basket.

9: Once the block is released from the mold, remove the fine layer of resin covering the sapphire disc.

10: Lift the sapphire with the tip of the razor blade to separate it from the resin block.

11: Sample is ready to be placed in a universal specimen holder (16701761) for further trimming and sectioning.