In theory, all samples with thickness <200 µm can be cryofixed by high pressure freezing (Studer et al. - 2008). First step is to position the two half cylinders and the middle plate in the loading place of the high pressure freezing machine (Leica system) (Figure 2). Then, under a binocular, the live sample (concentrated cells or dissected tissue) can be loaded in one 3 mm gold carrier (type A) in the 200 µm deep well (Figure 2). The filling and assembly of the different carriers is schematized in Figure 2. The specimen carrier must be completely filled with an embedding fluid (e.g. seawater, culture medium with cryo protectant if needed, see 4-Notes) without any air spaces. The level of water needs to be controlled since overfilling or under-filling the carriers can lead to poor freezing results. Excess of water can be slowly removed by capillarity using a dental stick (absorbent paper points) under a binocular (surface needs to be flat). Then, one 3 mm HPF carrier in aluminum (type B) is placed with its flat side towards the sample. So, the space between two carriers is 200 µm. If you have a large sample/tissue, 6 mm carriers can be used. (Figure 2). Once the assembly of the two carriers is obtained, it can be transferred with thin tweezers and inserted into the loading place (the middle plate) of the HPF instrument (Figures 2 and 3). Alternatively, Type A carrier can be already placed into the middle plate before sample loading. Manipulation of the sample should be as fast and gentle as possible, to avoid any damage and physiological stress of the samples.