I have immunoflorescence stainings done and some times for few antibodies, I had to give exposure of 600ms or above to detect the signal. what could be the reason ?
Thanks in advance
I have used 1.5 micron thick sections, could the thickness be the reason?
There is a variety of reasons why it make not be giving you good signal. It depends on the tissue you used and what antibody you're using. Is it one specific for that tissue? How long are you incubating the slides? At what concentration? What serum are you using to block? Are these cryosections or paraffin?
For example, I often stain 10 micron thick cryosections for apoptosis, but that requires an two hour incubation with the fluorescent secondary for good signal at 150-200ms.
Thanks Bahaar!
it is paraffin fixed kidney tissue with 1.5 micron thickness
goat serun is being used for blocking
primary conc. was 1:200 and secondary 1:2000
primary incubation- cold room, overnight
primary is in rabbit, ab purchased from CST
ab is specific to the protein
and it gives good signal with simlar conditions in lung tissue
Hi
You might try increasing the 2ndary antibody concetration say 1:500 to 1:1000. Your protein might be less expressed in kidneys compared to lungs. Moreover kidney tissues are way more compact than lung tissues, therefore you need higher concentration of your antibodies. In my case, lung tissue sections used to get overstained compared to other compact tissue, your case might be vice versa. Good luck.
Dear Moumita,
Can you please let me know the tissue thickness needs to be different for lung and kidney tissue ? We are using same 1.5 micron for both.
Hi Aparamita
Its fine if you use same thickness for the two. You need to lower blocking or increase antibody concentration for the kidney ones. I used go for increased blocking, lower antibody conc for lungs. All the best.
hi Moumita,
I yet have not tried high antibody concentration but improved conditions for antigen retrieval and I got the signal...
In addition to suggestions above, you can try longer blocking time with goat serum. I've found the signal greatly enhanced and specific when I incubated my slides for ~1-2 hours at least; overnight with certain tissues.
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