A freezing microtome (CM3050; Leica, Wetzlar, Germany) was used to cut the brain tissues into 8‐μm‐thick coronal sections. The slices were fixed with 4°C precooling acetone for 10 minutes and incubated with Iba‐1 goat polyclonal antibody (1:150, Abcam) and Mincle rabbit polyclonal antibody (1:200; Bioss corporation, Beijing, China) over night at 4°C. Next, the slices were incubated with secondary antibodies for 2 hours: Alexa 488‐conjugated donkey anti‐rabbit (1:200; Thermo Fisher), Alexa 594‐conjugated donkey anti‐goat (1:150, Thermo Fisher). The cell nuclei were stained with DAPI (Life Technologies, Billerica, MA, USA). Olympus LCX100 Imaging System and ImageJ were used for obtaining images and images analysis, respectively.
Alexa 594 conjugated donkey anti goat
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-conjugated donkey anti-goat is a secondary antibody that binds to goat primary antibodies. It is designed for use in immunofluorescence, flow cytometry, and other applications that require detection of goat-derived antibodies.
Automatically generated - may contain errors
2 protocols using alexa 594 conjugated donkey anti goat
1
Immunostaining of Microglia and Mincle
2
Quantifying Microglial TLR4 Expression
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Coronal sections of brain tissue (8-μm-thick) were cut using a freezing microtome (CM3050; Leica, Wetzlar, Germany). The slices were fixed with 4 °C pre-cooling acetone for 10 min, then anti-Iba-1 (1:150, Abcam, Cambridge, UK) and TLR4 (1:200, Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies overnight at 4 °C, followed by a 2 h incubation with secondary antibodies: Alexa 488-conjugated donkey anti-rabbit (1:200, Thermo Fisher, Waltham, MA, USA) and Alexa 594-conjugated donkey anti-goat (1:150, Thermo Fisher). Cell nuclei were stained with DAPI (Life Technologies, Billerica, MA, USA). The number of TLR4 or Iba-1 positive cells was calculated.
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