Hello,
I want to state that I have limited training on staining with alexa fluor 488.
I currently have to detect CD31 for angiogenesis on mice sectioned tissues. And I bought alexa fluor 488 anti mouse CD31 from Biolegend.
For the most part I know I need to block the tissues (5%BSA), and then incubate tissues with alexa fluor 488, followed by Dapi incubation.
But I don't know what concentration to start with. The alexa comes as .5mg/ml. I normally used 20uL in 1mL for in situ zymography staining.
Also, if anyone could recommend how to quantify the amount of protein/fluoresence for my images, that would be great.
Thank you for taking your time to answer,
You should do titration staining (1-5ug/ml) using your tissue sections.
I agree with Guozhong Qin, but if you do not want to play with dilutions, try 1:250. It should work.
I use dilutions of 1/200-1/400 (the first time i recommend you 1/200) it is very important the efficiency of the primary antibody and others factors...For quantify the amount of fluorescence: ImageJ is perfect!
Good luck!
Thank you for your answer. I went and tried with 1:100 dilution ( similar with in situ zymography test that I used). And the images came out good, some of areas more green fluoresence than the rest. But there is more signals in the lung tissues that I used as positive control. Also, I learned a way to quantify the signal by ImageJ. And to compare between samples, I calculate the flourescent/pixel/cell among different samples. Is that a good way to do so?
I have tried wth 1:100 (ABCAM overnight) and revealed with FITC anti mouse (Sigma 1.32 2 hours at 37). The results are very good! Stefano
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