I have differentiated the Neuronal cells. I used the PDL coated chamber slides for staining but these cells are vary loose attached with surface so I am getting lesser no of cells at the time of microscopy. I have tried to fix the neuron with 4% PFA for 20 min, 40 min, and 60 min but still neurons are detaching during washing.
Any suggestion to improve Fixing or is there any chemical to fix Neuron cells?
Thanks.
Cells in culture don't need a long time to fix. So you can shorten the fix time significantly. Also your cells are loosely adherent need to be treated gently. This may be your biggest problem rather than the fix per se. Can you increase the adhesion without disrupting the differentiation? Can you increase the PDL concentration or another adhesive substrate?
You should be able to achieve perfectly good fix in less than 5 min. The choice of fixative is really determined by the antibodies you are going to use for IHC. In general, light fixation is best for most antibodies but this can be determined empirically. I would suggest 2% PFA in PBS (cold) for 5 min. You need to add and remove the media and fix gently - tip the slide and draw off the medium from the side of the chamber. Wash twice. You can try fixing in culture media - but leave out the serum from the media - when you do this.
Obviously, glutaraldehyde 50% is stupid as commercially available is 25%. It is used from 0.5 to 2.5% only in suitable buffer. PFA also can be used 1-4% in buffer. Buffer must be 0.2 M. Time might be too long and shrinks the cells, try 5, 10, 15 min maximum. PFA penetrates fast enough, GA much slower. Depending on purpose, you can fix in mixture of GA and PFA. Wash gently with PBS
Cells in culture don't need a long time to fix. So you can shorten the fix time significantly. Also your cells are loosely adherent need to be treated gently. This may be your biggest problem rather than the fix per se. Can you increase the adhesion without disrupting the differentiation? Can you increase the PDL concentration or another adhesive substrate?
You should be able to achieve perfectly good fix in less than 5 min. The choice of fixative is really determined by the antibodies you are going to use for IHC. In general, light fixation is best for most antibodies but this can be determined empirically. I would suggest 2% PFA in PBS (cold) for 5 min. You need to add and remove the media and fix gently - tip the slide and draw off the medium from the side of the chamber. Wash twice. You can try fixing in culture media - but leave out the serum from the media - when you do this.
I agree with William J Brunken; cultured cells don't need a long time to fix. For fixation, you may try 4% PFA in PBS for 5 -10 min (4oC) and then postfix cells in absolute ethanol (-20oC) for 30 min. By this way, I found it significantly reduce the detachment of cultured neurons. Also whenever you apply any solutions on your cells, do it very gently. Good luck.
@ WIlliam J Brunken, Sir I agree with you that no need to fix the cells for long time, I did that for just to check wheather my PFA was working or not. Usually I do fixing only for 20 min. PDL concentration I am using is 100ug/ML, I cant increase the PDL concentration because the higher conc may cause toxicity to neurons. PBS using for washing is also fresh prepared but I am using it on room temperature. The handling was also so gentle but still I am facing the same problem. My staining is good so I dont think antibody is causing any problem here.
@Vipavadee Chaisuksunt, Mam thanks for your valuable suggestion I will try to postfix the cells with absolute ethanol then I will let you know my result.
Thanks Again.
Dr. Brunken (Hi, Bill. Hope all is well) is on the mark with being able to use short fixation times for cell cultures, and being ***very*** gentle with buffer exchanges. NEVER run liquids across the coverslips or culture dishes/slides (sometimes called "jet washing." This will do exactly what a jet engine would: Blow everything right off the coverslip.)
Another thing that may help is using buffers that contain Ca++, which is important to the function of a number of adhesion factors. We generally use Hank's Buffered Saline Solution, which contains physiological levels of calcium, for our preparations. If you normally use a PBS formaulation that doesn't contain Ca++ you can add CaCl2 with stirring to the PBS with stirring up to 1.2 mM final concentration.
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