Hi there,
I am doing immunohisto-staining and all my results came negative.. I washed the sections with 200 microliter of PBS. and now I am thinking that I should have immerse them in PBS rather than just adding 200 to the section.. which way is better?
Thank you in advance for answering
Hello Delilah,
In my case I wash the tissues with 200-500 ul of PBS for three times. Whatever the washing buffer I repeat 3 times and my results are pretty good. If you didn't now you can do that.
Best wishes.
If you are using on frozen sections, than you can use the way you are using, you can make circle with wax pen around. Frozen sections are more likely to come off
If you have paraffin section, I wash 4 min each by passing through PBS in 3 or 4 coplin jars which are filled with 80% of PBS, you can wash many slides at a time. If you have few slides, you may use slide mailer to conserve solutions
Any question, pl let me know
You can wash with PBS 3 times. If your laboratory don't have auto washer you can use DW pot .
Thank you
Use 0.01 M PBS + triton X -100 (0.4%) with a little pinch of glycine in it ( 3 times X 2 min in each step).
Try immersing them in the PBS solution for the required washing time rather than performing on the slide washing. Also make sure your antibodies are still ok to use.
Hi Tapas, what is the purpose of adding glycine to the washing buffer?
To remove any traces of aldehydes (that interfere with antibody binding) left in the tissue sections.
@Adrienne: more about you can find in already posted Q&A in RG, using the (full text) search function (top menu line, right to "JOBS" find the rectangle with the icon of a "person", a down-pointing arrowhead and "SEARCH". leftclick the downpointing arrow head (pull down menu) find and click QUESTIONS and insert your keyword/search phrase into it, e.g. " IHC protocol wash aldehyde " .... Then click the loop or "return", thus creating the/an URL: https://www.researchgate.net/search.Search.html?type=question&query=IHC++protocol++wash++aldehyde and some second later you'll find a lot of results in still existing Q&A. Best wishes and good luck WM
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