How to avoid salt crystallization in Immunocytochemistry?

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How to avoid salt crystallization in Immunocytochemistry?

Postby newbie17425 » Jan 20 2018 11:46 pm

I did an ICC and while imaging, I found that there are salt crystals on the coverslips. How can I avoid it?
My last steps are as follows:
After secondary, I wash the coverslips with PBS 3 times for 5 min.
I put a drop of my mounting media on a glass slide.
Take out the coverslip from the PBS, gently tap it on a kim wipe and place it on the glass slide.
Next, I keep the slides overnight at RT in dark and the following day I seal them using nail varnish.
Thanks
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Re: How to avoid salt crystallization in Immunocytochemistry

Postby mdfenko » Jan 21 2018 6:45 pm

are you performing the reactions on the coverslips?

if not, then why wash with pbs?

if so, then give a final rinse with either phosphate buffer (without salt) or water.
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Re: How to avoid salt crystallization in Immunocytochemistry

Postby newbie17425 » Jan 21 2018 6:47 pm

mdfenko wrote:are you performing the reactions on the coverslips?

if not, then why wash with pbs?

if so, then give a final rinse with either phosphate buffer (without salt) or water.


Yes, I am doing it on coverslips. After secondary ab, washing 3x with PBS, should I do an additional wash with 3x with water?
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Re: How to avoid salt crystallization in Immunocytochemistry

Postby mdfenko » Jan 21 2018 6:53 pm

just a quick rinse should be sufficient to remove salt. you don't want to expose the antibodies to the water for a prolonged period. you could use phosphate buffer (the same concentration that you use in the pbs).
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