Hi I am trying to make slides for IF imaging of DNA damage response proteins in HT1080 cells. When I observed the cells, I lost ~60%-70% of cells… only few cells are stained in cover slip. Rest of the cells are washed off! So loosing cells during preparation is my main problem!
Here the major steps are… Lyses the cell membrane by treated with Pre-Extraction buffer 2 mins on ice (1x PBS, 3 mM MgCl2, 300mM Sucrose, 0.2% Titron-X-100). Fix the cells in 4% PFA in PBS for 10 min at RT. Wash cells 2 x with PBS. Permeabilize the cells in 0.5% Triton-X 100 in PBS for 5 min at RT. Blocking with 1x PBS + 2% FBS for 1 hour followed by 1° ab and 2° ab – DAPI. Finally mounted in Prolong Gold Antifade reagent.
I observed that lot of cells are coming out as patches after (Permeabilization) Blocking!
Any suggestions would be welcomed! Thanks in advance!
I also never use the pre-extraction buffer you mention. I fix as you do, before lysing with PBS-0.1% triton 5 mins. I also don't bother with the blocking step. And keep the antibody incubations short too- 30-45 minutes. If this helps your cells stay stuck but you don't get good immunofluorescence, you can start extending incubation times to find a compromise between good staining and stuck cells!
If your cells are not very sticky, then you can coat the coverslips with poly-lysine solution before seeding cells.
PFA should fix the cells on coverslips. Try using freshly prepared PFA and/or coating the coverslips as suggested by O'Donnell. Lowering TX100 in permeabilization buffer to 0.2% or omitting this step completely may be tried.
I use 3% paraformaldehyde to fix my cells for a minimum of 10 min. I then wash in PBS (3x5 min), permeabiliise in 0.1% Triton-X-100 (10 min) before blocking for 1 h, all at room temp. Add the primary Ab for 1 h at room temp and the secondary can be done for 1 h at room temp or 4C overnight. Be careful of the density of the cells as if they are too confluent they may detach from the dish. Additionally, sudden temperature changes can also cause the cells to detach. Try to make sure that reagent etc are a similar temperature to the cells (e.g. room temp). What DNA damage process do you do? For example, if irradiating using a laser microbeam, then the laser power is too high. This may also affect the cells and their ability to remain attached. There are a number of things you can use to pre-treat your slides for better adherence such as poly-l-lysine, cell taq etc but in my experience this does not sufficiently improve cell adherence for this kind of issue.
Yes, I tried with methanol fixation, it was much better than that using PF.
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