Please take note of the general remarks below before using:
the post embedding immuno incubation protocol using Ultra Small Reagents
the post embedding immuno incubation protocol using Conventional Reagents
Recommended Incubation solution
PBS (10 mM phosphate buffered saline)
0.1 – 0.2% AURION BSA-c
check the pH and adjust to 7.4 if necessary
Buffers are either prepared immediately before use
or thawed from aliquots stored at -20°C.
Labeling ultrathin cryosections
After transfer of sections to a (nickel) grid first the covering sucrose layer has to be removed. If immuno incubations are performed immediately after sectioning the grids are simply rinsed on a drop of the incubation buffer. Mostly, however, grids are collected on a solidified 2% gelatine layer in buffer (corresponding to the incubation buffer) in a small Petri dish on ice. The sucrose layer which is facing the solidified gelatine is in this way allowed to diffuse gently away and to be at least partly replaced by incubation buffer. This procedure is supposed to be less destructive to the ultrastructure since concentration shocks are avoided. In this way sufficient grids can be collected and stored at 4°C if necessary overnight.If the grids with the sections were stored on a gelatine layer the closed Petri dish is warmed to 37°C for 30 minutes in order to liquefy the gelatine. Incubation buffer (37°C) is added in a 1:1 ratio to the liquefied gelatine.
On-grid labeling for electron microscopy
The use of nickel grids is recommended, especially if silver enhancement procedures are intended.
For most applications grids are floated on top of drops of immune reagents displayed on a sheet of Parafilm™. They are washed on larger drops of buffer. Whenever larger series of grids or coated grids need to be processed, the use of microtiter plates is preferred during incubations to avoid the risk of cross-contamination (e.g. Falcon 3034, Falcon Plastics, Oxnard, CA 93030, USA).
Transfer of the grids from droplet to droplet or from well to well can be performed with fine forceps. Preferably, a metal loop of 3.2 mm initial diameter made of nickel-coated copper wire of 0.2 mm thickness is used. The loops are flattened between flat beaks of a vice. The use of such loops diminishes the risk of contamination and greatly facilitates transfer of grids.
Procedures for marking
Procedures can e.g. be found in the CRC Press-edition “Immunogold Labeling in Cell Biology”, A.J. erkley & J.L.M. Leunissen eds., (1989), Boca Raton, Florida.In addition the issues of the Academic Press edition “Colloidal Gold”, M.A. Hayat ed., (1989), San Diego, California are highly recommended.