Hello,
I have a question about a mistake I made during my immunofluorescence protocol. I usually wash my coverslip in Triton X-100 0.1% in PBS before incubation with the secundary antibodies. The mistake is that I also made the last wash, before mounting, in Triton X-100 PBS 0.1% instead of PBS1x. Will this affect my experiment? Thanks to anyone who would help me.
Hello Chiara,
Personally, I don't think it will affect the result of the experiment.
Once I finish with the washes I usually wash the cover slips with DW and later I mount them.
Hope it was helpuful,
Nerea
Hello Chiara,
Triton x-100 is a detergent that with longer incubation could destroy too much the tissue even the antigens.
Also triton can increased the background fluorescence in the tissues mostly in lipid content attaching itself there.
I will recommend you to check your slides if you have them already stained and the best change your protocol and wash the slides with PBS.
Hopefully I help you :)
Hi,
What kind a problem do you have? Do you have a signal after staining? Is the background is too high?
Hello Justyna. Yes I have signal. I attacha photo. Tell me what you think about. Thanks
Hi,
Try to use a higher concentration of the primary antibody, anti-PERK.
You should improve the image quality.
You can use the serum (goat serum if secondary antibodies are a goat) before applying a primary antibody to eliminate non-specific antibody binding.
Can you show photos separately for Alexa Fluor 488, Alexa Fluor 546, nucleus dye?
Which protein was stained with Alexa 546?
I do not think your mistake affected the result in case it was just a brief wash.
It is difficult to tell anything about the quality if you do not know what protein was targeted with primary antibodies. green fluorescence is normally much weaker, so if you are using the confocal, to my mind, you need to increase the gain.
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