看完<雨天炎天&g
COCO李玟 1994-
前一阵子总算把一直做不出来的微管染色搞定了一些,最后还是buffer的选择决定成败,和当时的微丝的染色一样.参考了多篇文献后,最后New Techniques Enable Comparative Analysis of Microtubule Orientation, Wall Texture, and Growth Rate in Intact Roots of Arabidopsis这篇文献中的buffer和方法很好的起了作用,虽然他是用拟南芥为材料,不过在我的材料中也能工作.
不过微管的染色终究比微丝复杂,在一抗二抗这些方面就要花好多时间,现在看来一抗和二抗都过夜比较好,有次按照方法二抗没过夜,反而效果不好.
现在碰到的问题是这一方法仍然有局限性,只在根部的一部分能够染上,而根尖部分没有信号,我估计仍然是渗透不好.想到要推广应用至叶和茎尝试,估计也会碰到这个问题,到时试了再说吧.
最近打算把根部细胞同时进行微管和微丝的染色.看是否能看到两者之间联系的信号.不过一抗和二抗不多了,由于微丝染剂用的进口的molecular probe公司的,打算不知道能否微管的染剂也订些进口的.
引用
Whole seedlings were fixed in 1.5% (v/v) formaldehyde and 0.5% (v/v) glutaraldehyde made up in PEMT buffer(50 mm PIPES, 2 mm EGTA, 2 mm MgSO4, 0.05% [v/v] Triton X-100, pH 7.2) for 40 min and rinsed in PEMT buffer three times for 10 min. They were subsequently digested with 0.05% (w/v) Pectolyase Y-23 (Kikkoman) in PEM buffer (50 mm PIPES, 2 mm EGTA, 2 mm MgSO4) with 0.4m mannitol for 20 min, rinsed in PEM buffer three times,
treated with 220°C methanol for 10 min, and rehydrated in phosphate-buffered saline (PBS) for 10 min. Autofluorescence caused by free aldehydes from glutaraldehyde fixation
was reduced with 1 mg mL21 NaBH4 in PBS for 20min, followed by the treatment with 50 mm Gly in PBS(incubation buffer) for 30 min. Seedlings were incubated with primary antibodies against tubulin at room temperature overnight, rinsed in incubation buffer three times for 10 min, and secondary antibodies applied for 3 h at 37°C.After rinsing in PBS three times, root tips were cut off from the rest of seedlings, and mounted in 0.1% (w/v) paraphenylene diamine in 1:1 PBS-glycerol, pH 9. Cut cover glasses were used to space slide and cover glasses so as to avoid crushing delicate root tips.
treated with 220°C methanol for 10 min, and rehydrated in phosphate-buffered saline (PBS) for 10 min. Autofluorescence caused by free aldehydes from glutaraldehyde fixation
was reduced with 1 mg mL21 NaBH4 in PBS for 20min, followed by the treatment with 50 mm Gly in PBS(incubation buffer) for 30 min. Seedlings were incubated with primary antibodies against tubulin at room temperature overnight, rinsed in incubation buffer three times for 10 min, and secondary antibodies applied for 3 h at 37°C.After rinsing in PBS three times, root tips were cut off from the rest of seedlings, and mounted in 0.1% (w/v) paraphenylene diamine in 1:1 PBS-glycerol, pH 9. Cut cover glasses were used to space slide and cover glasses so as to avoid crushing delicate root tips.
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