I'm in the process of setting up IF staining for 5-methylcytosine (clone 33D3) and 5-hydroxymethylcytosine (the ActiveMotif polyclonal). Most protocols (and the manufacturer's instructions) use PFA fixed cells. However, in my hands these antibodies work only with acetone fixed cells, while especially the anti-mC antibody doesn't work at all with PFA fixed material. I'm slightly worried about this although the acetone fixed cells look good. Any experience with this? I'm doing cytospin preparates of human cells and yes I have the HCl denaturation step in the protocol.
That's a very good point. I don't have proper controls in my hands in the moment. So far I only know they both stain the nucleus (I could do a dot blot with methylated and unmethylated DNA, but of course that wouldn't be the same thing as whole cells). This is why I'm a bit worried that the staining doesn't work exactly according to the protocols.
Hi Mikael,
I do not have any experience with the antibodies in question, but have standardized a few immunofluorescence protocols. I have found PFA to be the best fixating agent besides its high toxicity. Due to this high toxicity it is very important to properly wash off the PFA. I also had trouble with antibodies and found that the fixation and permeabilization technique for the spesific antibodies were the real problem in my case.
Here is a staining protocol that works for most mammalian cells (MA104, Cos7, BSR, HEK, HepG2) I have used.
Fixating cells (24 well plate)
• Cells are fixated with 4% Formaldehyde
• Make up 4% Formaldehyde in phosphate buffered saline (PBS)
• Wash each well with PBS (~400ul) twice to remove any media
• Add 300ul of Formaldehyde to cells and incubate for 30 min at room temp (RT)
• Remove Formaldehyde and wash the cells at least three times with PBS to remove ALL traces of Formaldehyde
• Air dry cells (usually over night)
Rehydration and Permeabilization
• Rehydrate cells by adding 400ul of PBS and incubate at RT for 10 min.
• Make up an 0.25% Triton X solution in PBS (MP40 works as well)
• Add 300ul 0.25% Triton X solution to each well and incubate for 10 min at RT (preferably rocking gently)
• Wash the wells three times with 500ul of PBS (ensure all traces of Triton X is removed)
Preparation of cells for VP6 staining
• Rabbit specific HRP/DAB detection kit (ab64261) used
• Add 3-4 drops of H2O2 and incubate for 30 min at RT to block endogene peroxidase (preferably rocking gently)
• Wash the wells five times thoroughly with 500ul of PBS
• Add 3-4 drops of protein block and incubate for 30 min at RT (preferably rocking gently)
• Wash the wells five times thoroughly with 500ul of PBS
Primary and secondary antibody binding
• Prepare primary and secondary antibody binding as you would normally do
Hopes this helps...
Good luck
Proliferating or cultured cells normally have low levels of 5-hmC. Maybe below the detection limit? As control you could use primary tissue sections. Human gut has the proliferating crypts with low hmC and differentiated epithelium with higher hmC. Brain is the best positive control. Can also compete the staining with hmC rich synthetic oligos.
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