Hello, I've been comparing intracellular staining protocols and noticed that there are several alternatives for fixation with paraformaldehyde, varying time and concentration. Also, I read that increased PFA concentrations may affect some antigen's staining.
What do you know about? What are the optimal conditions?
Thanks for reading,
Hi Martin, There is no optimal time and PFA concentration for all antigens.
I agree with Yasaman on the %PFA and choosing antibodies that recognize epitopes after PFA fixation and potential background problems.
But if you are looking for small intracellular cytoplasmic proteins (not cytoskeletal actin, cytokeratin, etc.) you should include a detergent like digitonin in the PFA to permeabilize the cell membrane and fix the proteins in place.
Hi Martin, there is always to add something more or less specific when a subject is asked for quite globally / unpecifically. It seems that you might be a novice on RG and don't know about the possibility of searching the RG-database/platform (in case you did'nt know - see below- otherwise- apologize....(:-)).... For now I agree with the opinion and substantial relevant informations given by Yasaman A. and Steingrimur S. -
Just for further reading on the matter you were asking, you can try to SEARCH in the Q&A-threads which are saved in the RG-Database: If you do not know how to search in RG either find the ==> rectangle next right to JOBS in the RG-page - top menue line, click the second symbol in the rectangular window (next right to the sketch of a symbolic person - with the single left mouse click you open a pull -down menu for: #Researchers, #Publications, #Jobs, and #Questions (click there and write your keywords, e. g. formalin formaldehyde PFA FA fixation ) or copy and PASTE the resulting URL-notation:
https://www.researchgate.net/search.Search.html?type=question&query=formalin+formaldehyde+PFA+FA+fixation or insert e.g. other keywords you might search after:
| fixation formaldehyde intracellular staining | , resulting in an URL-notation:
https://www.researchgate.net/search.Search.html?type=question&query=fixation++formaldehyde++intracellular++staining ,
and you will be able to find some answer from the already given replies to more or less specific question.... Best of luck, WM
In in my lab we use 3,7% Formaldehyde to fix cells usually 10 min fixation but I notice that 3 to 5 min is better when you look about some rare or too sensitif antigens. To visualise intracellular proteins or intracellular part of membrane proteins (check for antibodies epitope) you must use detergent ex. Triton 0,01 or 0,02% for 2-3min after washing PFA or Formaldehyde.
If you are looking for soluble intracellular proteins this method is the best. If you're interested from cytoskeleton proteins you can try acetone or methanol fixation. This method fix and permeabilise no need detergent use. But because is permeabilising in the same time the soluble proteins are washed out.
If you are looking for intranuclear proteins you can use PFA/ Formaldehyde protocol but you must increase permeabilising time or even mix 0,01% Triton in PBS with your antibodies in this case is better to stain O/N in the fridge.
Staining in tissues increase background but you can use Sudan black to reduce.
You can also combine PFA (3.75% for 10-15 minutes) for fixing cells followed by methanol (5mins at -20°C) for permeabilizing cells. It works well, but depending on what your are looking for!!
It might be interesting to use microwave -aided fixation. I have used it for small tissue pieces and cell cultures , and the fixation time could be as little as 4- 10 seconds, depending on the samples. We found excellent structural preservation ( TEM), superior to immersion fixation. We have used it successfully for ultrastructural enzyme cytochemistry as well. There is a good deal of published info on the microwave fixation protocol to be found from literature search. May be you could avoid detergents , methanol etc with this protocol. Worth giving a try.
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