hi
so i was told that wherever possible, it is best to use warmed reagents when doing immunocytochemistry staining steps. In the below steps, which reagents must be warmed? is it safe to say that after the fixing step onwards, it is ok to use reagents that are not warmed? i did immunofluorescence just now but only used warmed PBS in step 2. The paraformaldhyde that i use for fixing is kinda cold and i dont know if this causes the rounding of my MDA-MB-231 cells which are supposed to look kinda elongated.
1) remove media from 35mm plate that has cells growing on coverslips.
2) wash with warmed 1X PBS.
3) fix with 4% paraformaldehyde.
4) permeabilize with triton
5) Block with BSA
6) Primary antibody staining
7) secondary antibody staining
I don't think that you need to prewarm either PBS or PFA beyond room temperature in order to proceed with PFA fixation of cultured cells. Conversely, if you fix cells or tissue sections with cold methanol or acetone, then it might be a good idea to pre-rinse cells with cold PBS, before adding the cold fixative and putting cells at -20. So, none of the reagents above should be warmed beyond RT.
Hi Iwan,
I think that the main point is how long it takes for your fixative to work. In the past I used 4% PFA cold and I did not noticed major changes in the overall cell morphology. However if your cells were already stressed and "loosely" attached it could be that the addition of the cold fixative triggered the change. (off course is always a good idea to look at the cell before adding the PFA and after the fixation to see what happens).
To go back to your question, I do not think is necessary to warm up the reagents in the following steps, however remember that temperature matters. Cold PFA fix the cells slower compared to RT or at 37ºC warm PFA. The permeabiisation also changes if run at 4ºC, RT or 37ºC. So is important that you keep your procedure constant among the experiments.
Good luck
As Fabian mentioned above, try to be "gentle" during fixation: do not throw the media directly onto the cells but rather to the sides of the dishes or the wells. So you can avoid detachment due to your handling. Howevere, if previous stresses already induced detachment, then there is little you can do for this.
Dear Iwan,
I would never use the solutions beyong room temperature for IHC. Usually I use the cold PFA and rinse with cold PBS. It works.
Good luck
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