Cryosections of 20 μm were fixed in cold methanol for 10 min and blocked for 1 h with normal goat serum, followed by tissue permeabilization in 0.1% Tween 20 in PBS for 5 min. Then the sections were incubated overnight with rat anti-mouse CD31 (BD Biosciences, Catalog No. 553370; 1.25 μg/ml) or polyclonal rabbit anti-Aβ40 antibody (Agrisera; 0.5 μg/ml) at 4 °C, and incubated with Alexa-594-conjugated goat anti-mouse IgG or Alexa-488 goat anti-rat IgG for 1 h at room temperature.
NTE was performed in darkness according to the previously described protocol [19 (link)]. Following immunostaining, the sections were directly submerged in ILFORD K5 emulsion, air-dried for 2 h at room temperature and exposed for 2 weeks at 4 °C. The emulsion-covered tissue sections were developed according to the manufacturer’s instructions, dehydrated in an increased series of ethanol solution and mounted with Pertex mounting medium. The immunofluorescence and NTE stainings were imaged with a Zeiss Observer Z.1 microscope using the ZEN 2.6 software (Carl Zeiss Microimaging GmbH, Jena, Germany).
NTE was performed in darkness according to the previously described protocol [19 (link)]. Following immunostaining, the sections were directly submerged in ILFORD K5 emulsion, air-dried for 2 h at room temperature and exposed for 2 weeks at 4 °C. The emulsion-covered tissue sections were developed according to the manufacturer’s instructions, dehydrated in an increased series of ethanol solution and mounted with Pertex mounting medium. The immunofluorescence and NTE stainings were imaged with a Zeiss Observer Z.1 microscope using the ZEN 2.6 software (Carl Zeiss Microimaging GmbH, Jena, Germany).
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