c*********8
发帖数: 369 | 1 有个染色的问题向各位专家请教，最近做Golgi 抗体染色，不知道怎么回事， 在显微
镜下看细胞基本上全绿，感觉核也是绿的，很难看到golgin 的部位，我用的first
antibody is golgin-97(human),mouse IgG1, monoclonal CDF4 (anti-Golgi) –
UNCONJ, the second antibody is Alexa Fluor® 488 Goat Anti-Mouse IgG (H+L
)，在加一抗前，也用2%BSA和10% goat serum block 1hour, 在加二抗前也是充分冲洗
，不知到怎么会事，想问问大家有没有什么有效的protocol分享以下，非常感谢，我的
邮箱：c*********[email protected] |
n***w
发帖数: 2405 | 2 how did you fix and permeabilize your cells? |
c*********8
发帖数: 369 | 3 Thanks for reply.
High-quality 3.7% formaldehyde (diluted in buffer); incubate 15
minutes at room temperature. Rinse each sample in buffer 3–4 times for one
minute.
Permeabilize the cells in 0.2% Triton X-100 (diluted in buffer) for 5
minutes |
n***w
发帖数: 2405 | 4 Is this the first time you ran this assay?
Did you have a no primary control?
Did you optimize your primary dilutions? |
a*****2
发帖数: 175 | 5 We use GM130 antibody from BD Bioscience that works very well even in
chicken cells. Also I only use 3%BSA for blocking. Paraffin section doesn't
work for Golgi neither based on my experience.
+L
【在 c*********8 的大作中提到】
: 有个染色的问题向各位专家请教，最近做Golgi 抗体染色，不知道怎么回事， 在显微
: 镜下看细胞基本上全绿，感觉核也是绿的，很难看到golgin 的部位，我用的first
: antibody is golgin-97(human),mouse IgG1, monoclonal CDF4 (anti-Golgi) –
: UNCONJ, the second antibody is Alexa Fluor® 488 Goat Anti-Mouse IgG (H+L
: )，在加一抗前，也用2%BSA和10% goat serum block 1hour, 在加二抗前也是充分冲洗
: ，不知到怎么会事，想问问大家有没有什么有效的protocol分享以下，非常感谢，我的
: 邮箱：c*********[email protected]
|
c*********8
发帖数: 369 | 6 Hi,apple12,thank you very much for your kind reply. Could you send me the
whole detailed Golgi protocol you’re used for GM130 antibody staining?
Thanks. I do not think the product has some problem become someone in our
lab already succeeded in this way but he already left for three years. I can
not reach him now. By the way, I already check the BD Bioscience website;
there are a lot of GM130 antibody. Also santa cruz biotechnology, inc. have
similar product. I feel a little confuse. Could you recommend which one is
better? Which kind of catalog number do you use? Thanks again |
