Supplementary material captions

Table summarizing the procedures used for single or double immunolabeling

Fig. S1 Diagram of the freezing system used to generate freezing/thawing cycles

A: Power supply, providing direct current 12 volts (V), five amperes (A). Peltier module: thermo-electric cooler. USB relay: relay turning the circuit on and off through a computer program. The relay was connected to the computer by a USB plug. See text for details. Heatsink and fan were taken from a computer power supply. The Peltier module was glued on the heatsink with thermic glue. The sample (in red) was left in a small cup made of aluminum foil and filled with water.

Fig. S2 Effects of microcryodissection on the mouse brain

(a-c) Aspects of the neuropil (left side) or white matter (right side) in the cortex without microcryodissection (a), after 80 cycles (b) or after 320 cycles (c). The tissue was more dissociated and in a more regular manner after 320 freezing/thawing cycles than after 80 cycles. The effects of microcryodissection on the mouse brain were qualitatively similar to those observed in the human brain. Mouse brain. Haematoxylin-eosin stain. Scale bar 80µm.

Fig. S3 The senile plaque in the cortex without microcryodissection

Senile plaque. Cortex, human brain, scale bar 8 µm. To be compared with Fig. 5 (a) Aβ immunohistochemistry (6F3D). An extracellular Aβ deposit is concentrated in its center into an amyloid core (ac). (b) Phospho-tau immunohistochemistry (AT8). The neuritic crown (nc) is made of numerous and dense immunoreactive neuropil threads, surrounding the amyloid core. Without microcryodissection, the relationships between the amyloid core and its crown are elusive. (c) CD68 immunohistochemistry. A macrophage (blackarrow) is seen near the amyloid core. (d) Double labeling myelin basic protein (MBP) in brown and neurofilament (NF) in red. Myelinated axons (MBP positive) are seen around and across the senile plaque (arrowheads). Some NF positive profiles are seen within the the plaque (greyarrows). (e) Anti-synaptophysin immunohistochemistry. Immunoreactive dots, corresponding to abnormally large synapses (greyarrows), were diffusely seen within the plaque. (f) GFAP immunohistochemistry. The plaque was surrounded by GFAP positive fibers (arrowheads).

Fig. S4 Electron microscopy of a senile plaque, with or without microcryodissection

(a, b) Senile plaque without microcryodissection. Human brain, neocortex, scale bar 5µm. (a) Some macrophages (arrows) were observed in the vicinity of the amyloid core (*).(b) Numerous dystrophic neurites (arrows) were observed around the amyloid core (*). (c, d) Magnification of two dystrophic neurites (arrows). Some of those dystrophic neurites were seen in contact with amyloid fibrils (*). They contained dense bodies, occasionally laminated. Human brain, neocortex, scale bar 1µm (c) or 500nm (d). (e) After 80 freezing/thawing cycles, the components of the senile plaque were slightly dissociated (arrows for dystrophic neurites; * for amyloid core). Human brain, neocortex, scale bar 5µm. (f) After 320 freezing/thawing cycles, a neuron (arrow) was separated from the neuropil. Some details of the perikaryon are still visible. A blood vessel (arrowheads) was also isolated. Human brain, neocortex, scale bar 10µm.

Fig. S5 Microcryodissection of diffuse Aβ deposits in the striatum

(a) Microcryodissection of diffuse Aβ deposits in the striatum. Aβ immunohistochemistry (6F3D antibody). Scale bar 80 µm. (b, c) Magnification of the diffuse deposits, scale bar 8 µm. (b) Diffuse Aβ deposits were often found at a distance from any neuronal cell body. (c) The cell body of a neuron could rarely be trapped in a diffuse deposit. Such neurons did not contain any Aβ in their cell body.

Fig. S6 Aspects of tau pathology after microcryodissection in the human cerebral cortex

(a) Phospho-tau immunohistochemistry (AT8) in the neuropil. A classic AT8 positive neurofibrillary tangle (*) was seen in the cytoplasm of an affected neuron, next to another neuron devoid of immunoreactivity, except for a synapse-like dot at the periphery of its cell body (blackarrow). Phospho-tau positive neuropil threads appeared fragmented in the mesh of the neuropil. Scale bar 8 µm. (b) Ubiquitin immunohistochemistry in the neuropil. Ubiquitin positive neuropil threads were not fragmented but continuous, and wound along the fiber fascicles that formed the mesh. Scale bar 8 µm.

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Supplementary table

Tau-Aβ double labelling / MBP-NF double labelling / Simple labelling
Antibody / Anti-Phospho-Tau pS202/T205 (AT8) / Anti-Human Beta-Amyloid (6F3D) / Anti-Human Myelin Basic Protein (MBP) / Anti-Human Neurofilament Protein (NF) / Anti-Phospho-Tau pS202/T205 (AT8) / Anti-Human Beta-Amyloid (6F3D) / Anti-Human Synaptophysin / Anti-Human CD68 (CD68) / Anti-Human Glial Fibrillary Acidic Protein (GFAP) / Anti-Ubiquitin
Reference / MN1020 / M0872 / A0623 / M0762 / MN1020 / M0872 / M7315 / M0814 / M0761 / Z0458
Dilution / 1/500 / 1/200 / 1/2000 / 1/2000 / 1/500 / 1/200 / 1/50 / 1/1000 / 1/500 / 1/300
Clone / AT8 / 6F/3D / - / 2F11 / AT8 / 6F/3D / DAK-SYNAP / KP1 / 6F2 / -
Specie / Mouse / Mouse / Rabbit / Mouse / Mouse / Mouse / Mouse / Mouse / Mouse / Rabbit
Mono/ / Monoclonal / Monoclonal / Polyclonal / Monoclonal / Monoclonal / Monoclonal / Monoclonal / Monoclonal / Monoclonal / Polyclonal
Polyclonal
Provider / Thermo Scientific / Dako / Dako / Dako / Thermo Scientific / Dako / Dako / Dako / Dako / Dako
Procedure / Formic acid (5 min), Cell conditioning (CC1 solution, 30 min), primary antibody (anti-phospho-Tau AT8, 32 min), Secondary antibody (HRP multimers, 8 min), Detection (DAB kit), Denaturation (90°C, 4 min), primary antibody (anti-Aβ 6F3D, 2h), Secondary antibody (AP multimers, 12 min), Detection (Alkaline Phosphatase (AP) Red kit), Counterstaining (Haematoxylin II, 12 min), Post-counterstaining (Bluing Reagent, 8 min). / Cell conditioning (CC1 solution, 30 min), Primary antibody (anti-MBP, 32 min), Secondary antibody (HRP multimers, 8 min), Detection (DAB kit), Denaturation (60°C, 8 min), Primary antibody (anti-NF, 20 min), Secondary antibody (AP multimers, 12 min), Detection (Alkaline Phosphatase (AP) Red kit), Counterstaining (Haematoxylin II, 8 min), Post-counterstaining (Bluing Reagent, 4 min). / Cell conditioning (CC1 solution, 30 min), Primary antibody (32 min), Secondary antibody (HRP multimers, 8 min), Detection (DAB kit), Counterstaining (Haematoxylin II, 8 min), Post-counterstaining (Bluing Reagent, 4 min). / (Manually)
Formic acid (5 min), Primary antibody (overnight), Secondary antibody (1h), Detection (DAB kit), Counterstaining (Harris haematoxylin, 5-15 s), Post-counterstaining (H20, 5 min). / Cell conditioning (CC1 solution, 30 min), Primary antibody (32 min), Secondary antibody (HRP multimers, 8 min), Detection (DAB kit), Counterstaining (Haematoxylin II, 8 min), Post-counterstaining (Bluing Reagent, 4 min). / Cell conditioning (CC1 solution, 30 min), Primary antibody (20 min), Secondary antibody (HRP multimers, 8 min), Detection (DAB kit), Counterstaining (Haematoxylin II, 8 min), Post-counterstaining (Bluing Reagent, 4 min). / Cell conditioning (CC1 solution, 30 min), Primary antibody (28 min), Secondary antibody (HRP multimers, 8 min), Detection (DAB kit), Counterstaining (Haematoxylin II, 8 min), Post-counterstaining (Bluing Reagent, 4 min). / Cell conditioning (CC1 solution, 30 min), Primary antibody (32 min), Secondary antibody (HRP multimers, 8 min), Detection (DAB kit), Counterstaining (Haematoxylin II, 8 min), Post-counterstaining (Bluing Reagent, 4 min).

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