I'm having trouble with my nuclei clumping together, which makes it difficult for me to count them under the microscope. The protocol we use has us place the thawed tissue sample in PBS+PMSF and then fix the tissue in formaldehyde. After stopping the fixation with glycerine, we centrifuge the sample, remove the supernatant, and then resuspend the pellet in PBS+PMSF. From there we homogenize the tissue using needle and syringe. To count the nuclei, we add trypan blue to an aliquot of the sample and count the nuclei. Does anyone have an idea of what could be causing the nuclei to look like this? I'm not sure if the nuclei are just clumped together or if they're actually broken. Thanks in advance.
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tm1987
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Posted 06 September 2012 - 01:16 PM
Hi everyone
I'm having trouble with my nuclei clumping together, which makes it difficult for me to count them under the microscope. The protocol we use has us place the thawed tissue sample in PBS+PMSF and then fix the tissue in formaldehyde. After stopping the fixation with glycerine, we centrifuge the sample, remove the supernatant, and then resuspend the pellet in PBS+PMSF. From there we homogenize the tissue using needle and syringe. To count the nuclei, we add trypan blue to an aliquot of the sample and count the nuclei. Does anyone have an idea of what could be causing the nuclei to look like this? I'm not sure if the nuclei are just clumped together or if they're actually broken. Thanks in advance.
I'm having trouble with my nuclei clumping together, which makes it difficult for me to count them under the microscope. The protocol we use has us place the thawed tissue sample in PBS+PMSF and then fix the tissue in formaldehyde. After stopping the fixation with glycerine, we centrifuge the sample, remove the supernatant, and then resuspend the pellet in PBS+PMSF. From there we homogenize the tissue using needle and syringe. To count the nuclei, we add trypan blue to an aliquot of the sample and count the nuclei. Does anyone have an idea of what could be causing the nuclei to look like this? I'm not sure if the nuclei are just clumped together or if they're actually broken. Thanks in advance.
