Does anyone have experience with Ki67 staining on astrocytes, microglia or OPC's?
I have tried with Ki67 and DAPI staining, but the Ki67 staining is blurred, so I'm interested in how other have experienced it on these types of cells
Dear Emilie Olsen, how it is your blocking step? your cells are from mice or rat? Could you give us more details about antibodies brands and stainings steps like incubation time, blocking and antibodies solution?
Hi Markley Silva Oliveira Junior
I'm using cells from mice pups.
I'm blocking the cells after fixation for 1 hour with 1xPBS, 0,25% BSA and 0,25% Triton-x-100. After blocking, I use Ki67 from Abcam (ab15580), and I have tried 1:1000 dilution in blocking buffer - in the moment I'm trying with 1:500 - and then it is incubated overnight at 4ºC. The next day, I wash the plate in washing buffer, containing 1xPBS and 0,25% BSA for 3 times. Then the secondary is added (also diluted in blocking buffer). Here I have used FITC (1:600), from Immuno Jackson, but next I will try Alexa 488 (1:400). The secondary is incubated for 2 hours, and the plate is washed 2 times with washing buffer, and then stained with DAPI and 1 time of wash in washing buffer.
Hope that this is giving you a better understanding in my procedure - otherwise, let me know :-)
And thank you for taking the time to answer.
Hey, Hi Emilie! Anytime, my pleasure
You can make a double test, with/without DAPI to see, if your DAPI is causing these problems. How old is your DAPI solution?
If the problem persists, you can try the following steps,
-A good tip is a previous/preliminary block before the blocking step, just with 0.5% Triton x-100 for 1hour.
-Then, NGS10%, BSA1% and 0.5% triton x-100 both diluted in PBS, is a good solution for mice staining. This solution works quite good for me when I use Mcm2 and PCNA for cycling cells, mostly with neural stem cells/astrocytes progenitors from mice pups. I use this solution as blocking (for 2 hours) and as primary (overnight) and secondary antibody (2 hours) solution too.
-Ki67 could be used more concentrated, I strongly recommend 1:500 with this one from Abcam. In my opinion, 488 and 594 are the better ones for nuclei stainings, so you`re doing good at this. I prefer to use Alexa 594 Goat anti-something for Cycling markers on mice tissue. More washes (at least 3) between the steps will give you less background too.
Hope's this help you,
Good luck over there,
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