I am doing IF staining for intracellular domain of HER2 receptor. Sometimes I keep the cells in 4% PFA for 1-2 weeks in the fridge till I stain them. Does this affects the antibody biding affinity?
Hello,
Yes, it is likely to hamper antibody binding due to over cross-linking because PFA is a cross-linking fixative.
Usually, cells are fixed for 10 minutes at room temperature with 4%PFA in PBS followed by 2-3 washes with PBS to remove excess PFA and stop the fixing reaction. Fixed cells are stored in PBS at 4 degree C.
Over incubation in 4%PFA may be damaging as it may reduce antibody accessibility to the epitope.
Best.
All said above it´s true, to fix cells during 1-2 weeks is absolute savagery, and not only affect the epitope you want the antibodies to bind, it may cause a loooooot of background autofluorescence (green/yellow range).
Take care of your tiny little cells man, they are very delicate.
Best wishes!
If your antigen of interest no longer reacts with the antibody, try chemical antigen retrieval: https://www.abcam.com/protocols/ihc-antigen-retrieval-protocol
Malcolm Nobre Oscar Recasens Ellen A G Chernoff thank you so much for your valuable inputs and suggestions!
Hello Nooraldeen, yes, I found that a long postfixation of brains in PFA affects antibody binding, due to over cross-linking. It happened to me, expecially with nuclear antibodies (e.g. anti-ki67). For this reason, in my lab we prefer to do a short-time postfixation in PFA, non going beyond an overnight postfixation at 4°C.
My suggestion is to postfix the samples for 2/3 hours and then put the samples in PBS + Na Azide, until you put sucrose for cryopreservation and then freezing.
Roberta Schellino thank you for answering my question. Currently, I am doing 1-2 hours fixation and then 1%BSA (in PBS)+ NaN3 till I stain them and it worked very well.
No , I used formaline before with my cells and the rusult was perfact.
To achieve a high best resolution and immunoreactivity it is recommended to fix tissues with aldehyde-based fixatives, whereas the use of non-aldehyde–based fixatives can be advantageous in obtaining high protein yield.
Article The Impact of Tissue Fixatives on Morphology and Antibody-ba...
Of course, it will have an impact, and it is recommended to use 4% PFA for fixation for less than 15 minutes and then proceed directly to IF. A good immunoreaction will be obtained by you.
The first thing is to clarify the fact that paraformaldehyde has no fixative function because it’s a powder. The fixative solution is a formaldehyde. As the others wrote, long fixation is cross linking the epitopes. You should not fix the cells or tissue for more than 24h. Then to enhance the antibodies prnetration into the cell you can use triton X at 0.25% for a few minutes to permeabilize the cells membrane.
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