Alexa fluor 488 labeled donkey anti rat igg antibodies

After deparaffinization, slides were pre-treated in citrate buffer in microwave for 1 min and blocked with 10 % donkey normal serum for 30 min at room temperature. Primary antibodies anti-GFP, anti-CD31, anti-CD68 were each diluted at 1:100 with Can Get Signal (Toyobo Co., Ltd, Osaka, Japan) and allowed to react overnight at 4 ^oC.
For secondary antibody, Alexa Fluor 568 Labeled Donkey ant-goat Ig G antibodies (Life Technologies, Palo Alto, CA, USA) and Alexa Fluor 488 Labeled Donkey anti-rat IgG Antibodies (Life Technologies, Palo Alto, CA, USA) and Can Get Signal (Toyobo Co., Ltd, Osaka, Japan) were diluted at 1:200 and allowed to react for 60 min at room temperature. Then after, the nuclei were stained with 1 mg/ml of DAPI for 3 min. Slides were then washed with TBS and mounted using Fluorescent Mounting Medium (Dako Japan Co., Ltd, Tokyo, Japan).
The study conformed to the experimental guidelines in animal experiment of Matsumoto Dental University approved by the Animal Laboratory Examination Committees of the University (Approval number # 233-13).

After deparaffinization, slides were pre-treated in citrate buffer in microwave for 1 min followed by blocking with 10 % donkey normal serum for 30 min at room temperature. The primary antibodies used namely anti-GFP, anti-S100A4, anti-Runx2, anti-CD31, Can Get Signal (Toyobo Co, Ltd, Osaka, Japan at 100-fold) and were allowed to react overnight at 4 ^oC.
Secondary antibodies used were Alexa Fluor 568 Labeled Donkey Anti-Goat IgG Antibodies (Life Technologies, Palo, Alto, CA, USA), Alexa Fluor 488 Labeled Donkey Anti-Rat IgG Antibodies (Life Technologies, Palo, Alto, CA, USA) and Can Get Signal (Toyobo Co, Ltd, Osaka, Japan), diluted at 200-fold and reacted for 60 min at room temperature. DAPI 1mg/3ml for 3 min was used for nuclear counterstain. After washing with TBS, slides were mounted using Fluorescent Mounting Medium (Dako Japan, Co, Ltd, Tokyo, Japan).