For my stainings of 3 um thick slides of mouse liver tissue (fixed for 24 hrs at 4C, 4% paraformaldehyde) I accidentally didn´t use Triton X-100 to permeabilize cell membranes. I did it the same way for all of my samples. I want to label BrdU-positive nuclei from hepatocytes. All in all, it seems that I got good and reliable signals according to my positive and negative controls.
However, on the internet I only found protocols which use Triton X-100. What would be your recommendation? Do all my stainings again with Triton X-100 because apparently everybody is doing it this way or rather stay with my protocol for future stainings?
Thanks a lot for your answers.
Christian
As you know, Triton X-100 is for permeabilising the "intact" membranes in a cell so that the primary and secondary antibodies can get into the subcellular structure and combine to the target molecules. Usually a nucleus may have around 3 to 5 micron in size (depends on what kind of tissue you are studying). When you cut paraffin slices at 3, most of the nuclei might be sliced through. That is why you could get good staining without using Triton-X 100.
If you use 50-micron vibratome or frozen sections, triton should be necessary, generally.
Hi Christian
I think this article may helpe you
N Am J Med Sci. 2010 May; 2(5): 241–245. [Double staining immunohistochemistry]
doi: 10.4297/najms.2010.2241
Hi Christian,
Triton X-100 is useful to permeablise cells with intacted cell membrane, for instance those cells fixed in a chamber slide. However, with parafin fixed tissues, that might not always be required.
In my experience, if you have got good staining with good negative control, I believe that your method is good enough for your purpose.
I am not expert. Hope this helps.
As you know, Triton X-100 is for permeabilising the "intact" membranes in a cell so that the primary and secondary antibodies can get into the subcellular structure and combine to the target molecules. Usually a nucleus may have around 3 to 5 micron in size (depends on what kind of tissue you are studying). When you cut paraffin slices at 3, most of the nuclei might be sliced through. That is why you could get good staining without using Triton-X 100.
If you use 50-micron vibratome or frozen sections, triton should be necessary, generally.
Dear Christian,
When you are cutting 3 micron sections you are actually removing the plasma membrane from all your cells, and therefore you will not need the Triton x-100 procedure. I also think that the nuclear membranes contain a lot less lipids/more protein plus nuclear pores which seem to open up in dead/fixed cells, and therefore does not really require the harsh permeabilisation process. There were similar questions in the past related to nuclear staining and you may find my previous replies in the report below.
best wishes, Refik
Technical Report Research Methods and Technical Considerations: Answers to ov...
I agree with Refik Kanjhan, that when you have very thin section - you don't need Triton-Х-100, for example, when we prepare the wholemount of planarians - we use the Triton in preincubation solution for above 24-48 hours< and when we cut sections (15 mkm) - we only use twash them with the solution containing Triton 3 times per 5 min ( i.e. for shorter time), Good wishes< natalia
Agree with Xiong, no need to do it on less than 20 micron on the contrary it could cause false positive staining . I would not recommend it frozen but for paraffin section only.
Dear scientists,
Thank you all for your helpful advice. I appreciate it.
Best,
Chris
Since I accidentally stumbled across this really old thread, honestly (and also perhaps as an additional information for Özlem Mutlu) added:
If you are able / allowed to open AND search "Google" for the following keywords [copy and paste words within vertical lines/dashes ( = text/words in BOLD as displayed - including quotation marks) just only into the GOOGLE-, or Mozilla- Firefox - or any other Internet Browser] :
| "secondary antibody" washing AND "TRITON X-100" |
you'll get a lot (over 1.170,000) of informative articles (at least the first 20 results) dealing with the matter you wanted to know about.
If you want to know more about how to apply to vibratome sections, search for | "secondary antibody" washing AND "TRITON X-100" AND vibratome AND SECTIONS | and find at least with that modified search phrase:
a considerable fraction (31,400) of positive results (as compared to the first search phrase as given above) especially within the first 20-30 results.
Since it always depends whether or not to "use something / anything in processing for xyz" I don't want to say anything about pro's and/or con's in using Triton X-100 and washings.......It depends really....
NB: If you - for any reason - can't search by yourself for the keywords given above I would be able to propose or even send some references / literature or recipes sending a list to your e-mail address.
If such action is/would be relevant, either write via the ResearchGate-messaging system to me (in that system I don't have much possibilities to enclose literature-files) or send your request (for some results of the proposed "google search") to: [email protected].
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