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Immunohistochemical analysis

TS Tomoyasu Shiraya
FA Fumiyuki Araki
TU Takashi Ueta
HF Hisako Fukunaga
KT Kiyohito Totsuka
TA Takahiro Arai
AU Akiyoshi Uemura
KM Kyoji Moriya
SK Satoshi Kato
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Frozen 10-μm sections on slides were dried for 30 min at room temperature, rehydrated in PBS for 5 min, incubated with blocking solution (5% normal goat serum and 0.5% Triton X-100 in PBS) for 1 h, and then stained with primary antibodies for overnight at 4 °C overnight. The slides were washed with PBS three times for 10 min each time and incubated with secondary antibodies at 4 °C overnight32. We used a rat monoclonal antibody specific to F4/80 (Cl: A3-1, 1: 1000, Bio-Rad Laboratories, Inc. Hercules, CA, USA) for the primary antibody and an Alexa Fluor 488-conjugated secondary antibody (1:400), and the nuclei were stained with DAPI (1: 2000). The specimens were observed under a laser confocal microscope (LSM800, Zeiss, Oberkochen, Germany).

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