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asked in 科學生物學 · 1 decade ago

救救我'' 我需要詳解

Immunofluorescence Microscopy (THE JOURNAL OF BIOLOGICAL

CHEMISTRY VOL. 283, NO. 4, pp. 2454–2464)

After transfection, the cells were washed with phosphate-buffered saline and fixed with

4% formaldehyde in phosphate-buffered saline for 5 min at 25 °C followed by methanol

at -20 °C for 15 min. The fixed cells were probed with rabbit anti-hNinein serum (1:500,

our preparation), rabbit anti-Astrin antibody (1:500, our preparation), mouse anti-Astrin

antibody (1:500, our preparation), rabbit anti-Aurora A (1:500, our preparation), rabbit

antipericentrin (1:500, Abcam), mouse anti-BubR1 (1:250, BD Biosciences), mouse

anti-CENP-E (1:250, Abcam), mouse anti-tubulin antibody (1:1000; GTU-88, Sigma),

and mouse anti-tubulin antibody (1:1000; DM 1A, Sigma). DNA was stained with

4,6-diamidino-2-phenylindole (2 μg/ml). Immunofluorescent cell images were acquired

using an Olympus LSM Fluoview 500 Confocal laser scanning microscope (Olympus).

Images were processed with Adobe Photoshop software (Adobe Systems). All data were

collected during the same session to avoid potential differences due to fading. Images

were then measured for area and total pixel number for each image by circumscribing

spindle contours and selecting that region for quantitation. Pixel number was obtained by

multiplying the selected area by pixel intensity.




Update 2:





3 Answers

  • 嬌嬌
    Lv 6
    1 decade ago
    Favorite Answer


    螢光顯微鏡 (的期刊的生物化學 VOL.283、 號 4 頁 2454–2464) 後轉染,儲存格被沖洗磷酸鹽緩衝鹽和固定在 5 分鐘 25 C 後, 跟在 15 分鐘跌 C 的甲醇磷酸鹽緩衝鹽水的 4%甲醛。 固定的儲存格被探測兔抗 hNinein 血清 (1:500,我們準備)、 兔抗 Astrin 抗體我們製備 1:500)、 滑鼠抗 Astrin 抗體 (1:500,我們製備) 兔 anti-Aurora 一 (1:500,我們製備),兔 antipericentrin (1:500,Abcam) 滑鼠抗-BubR1 (1:250,屋宇署生物科學) 滑鼠抗 CENP-E (1:250,Abcam) 滑鼠 anti-tubulin 抗體 (1000 ; Sigma GTU 九八七) 和 (1000 ; 糖尿病 1A,Sigma) 的滑鼠 anti-tubulin 抗體。 DNA 是 4,6-diamidino-2-phenylindole (2 μg/毫升) 染色。 螢光細胞圖像被獲得一個顯微鏡 (Olympus) 的 Olympus LSM Fluoview 焦 500 鐳射。

    Adobe Photoshop 軟體 (Adobe Systems) 處理影像。 為了避免潛在的差異,由於褪色的同一個會話過程中收集所有的資料。 圖像,然後測量的每個圖像的地區和總圖元數 circumscribing 主軸輪廓並選擇該區域的定量。 圖元數獲得通過將選定的區域乘以圖元強度。

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  • Anonymous
    1 decade ago



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