Terry 發問時間: 社會與文化語言 · 6 年前

急急急~關於Immunochemistry的原理和圖形判讀

各為大大拜託幫小弟個忙 我在翻paper時看到這個方法Immunochemistry

我想知道他的原理和次級抗體含biotin與之後再用(受質)streptavidin-peroxidase complex培養後用3,3-diaminobendizine (DAB)染色後再用蘇木紫複染,然後DAB染色成棕色代表biotin和streptavidin有結合,那用蘇木紫複染幹嘛

整個流程有點看不太懂 拜託高手解救我>< 下面是這段是我paper利用Immunochemistry所做的過程

The expression of GRP and GRPR in

the afore-mentioned three cell lines was assessed by immunocytochemisty.

Cells plated on glass cover slips were fixed by

4% paraformaldehyde, and permeabilized with 0.5% (v/v)

Triton. Cells were washed with PBS, treated with ice-cold

methanol for 20 min, and pre-blocked for 30 min with sheep

serum at 37˚C. Cells were subsequently washed twice with

PBS, and incubated with anti-GRP and anti-GRPR (1:100)

overnight at 4˚C. After PBS wash, cells were incubated with

secondary goat anti-rabbit immunoglobulin conjugated with

biotin for 60 min at room temperature, followed by incubation

with streptavidin-peroxidase complex for 30 min. The

peroxidase reaction products were visualized by incubation

with 0.05 M Tris-HCl buffer (pH 7.6) containing 20 mg

3,3-diaminobendizine (DAB). The slides were counterstained

for nuclei with hematoxylin and mounted with gum.

1 個解答

評分
  • Beth
    Lv 7
    6 年前
    最佳解答

    整個流程依序如下:

    把細胞放到玻璃蓋片上後, 將細胞

    1. fixed by 4% paraformaldehyde

    2. permeabilized with 0.5% (v/v) Triton

    3. washed with PBS

    4. treated with ice-cold methanol for 20 min

    5. pre-blocked for 30 min with sheep serum at 37˚C

    6. washed twice with PBS

    7. incubated with anti-GRP and anti-GRPR (1:100) overnight at 4˚C

    8. incubated with secondary goat anti-rabbit immunoglobulin conjugated with biotin for 60 min at room temperature

    9. incubation with streptavidin-peroxidase complex for 30 min

    顯現peroxidase reaction的產物的方法:

    10. incubation with 0.05 M Tris-HCl buffer (pH 7.6) containing 20 mg 3,3-diaminobendizine (DAB)

    11. The slides were counterstained for nuclei with hematoxylin ,然後用膠(gum)將載玻片(slides)固定起來.

    參考資料: 剩下的都是專業的用語
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