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enter image description hereenter image description hereI am using the same protocol and same antibodies that the literature says but still I cannot get good staining for my lymph node sections, I tried to change the fixation method and I am using now acetone instead of PFA, I also tried with acetone plus ethanol, the antibody concentration used worked in previous sections. How could I increase the signal and/or remove the noise?

It also happened to work in some sections before but not in others even I did exactly the same, why could that be if I am using the same tissue?

Thanks

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    $\begingroup$ could you upload example of microscopic image $\endgroup$ – aaaaaa Jun 19 '18 at 21:14
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    $\begingroup$ Is the tissue you are staining now the same age (i.e., time from initial fixation to staining) as the tissue you used before? $\endgroup$ – Bryan Krause Jun 19 '18 at 22:38
  • $\begingroup$ yes I do the fixation just before the staining so I treat them in the same way $\endgroup$ – Bio Jun 20 '18 at 12:41
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    $\begingroup$ Did you try a different fluorophore or fresh antibody? What're you using now? And are you getting signal with other markers like DAPI? What does your staining efficiency look like by FACS? Strong separation from negative or weak? Maybe try digesting some lymph node and see if you get good staining. $\endgroup$ – CKM Jun 20 '18 at 17:35
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    $\begingroup$ @bio what's your red laser filter setup? $\endgroup$ – CKM Jun 29 '18 at 2:59

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