Hello,

I've used 0.1 M NaP, pH 7.4 containing 4% paraformaldehyde when carrying out TUNEL staining; I left it at room temperature for a number of weeks and it worked just fine.

Christopher

Hi, Florence, you can dissolve para-formaldehyde in water with gentle warming up to 70°C. You may add small drop of NaOH for better dissolving.

I would suggest you to prepare 4% solution in water. There after you have to mix it with equal amount of 2XPBS (or MTSB or simple NaP, all at pH 7.0). You can keep stock at RT for short time (1-2 week), but, before usage, you have to check presence of the pellet. If you do not have pellet, you can use it.

Good luck!

Formaldehyde depolymerizes at room temperature and when exposed to air to form formaldehyde gas. It can be stored at 4C for a week or 2 for best results or aliquot and store at -20C for long term storage (several months). With almost anything stored in aliquots at -20C, avoid repeated freeze/thaw cycles. Now it is also dependent on whether it is made in a buffered solution. If made in just water, the pH will change faster and you should use it after making it fresh.

If making it in a buffered solution, it is easier to dissolve the paraformaldehyde in water by heating to maximum 80C, then dissolve any leftover PFA with some drops of NaOH. Then add a 2X concentrate of your buffered solution (dissolving directly into a buffered solution may not be as efficient). If you do store for a long time, be weary of any precipitates that form.

I have used chilled 4% paraformaldehyde, in PBS to fix HEK cells, approximately 10 minutes incubation time. Also, I would do the incubation on ice. Wash the cells with chilled PBS 3 times before adding methanol. Freshly made paraformaldehyde is always better but stored at 4 degrees C, it should be fine to use.

Thanks everyone for your suggestions.

At 4% this is a very strong fixative.

A 1,5% solution is more gentle and can perhaps preserve finer structures in a better way.It may be worth a try.

It all depends on your requirements of your fixed cells and further investigations.

Often a two step fixation is preferred with formaldehyde that penetrates faster but fixes less firmly, followed by paraformaldehyde but if you fix single cell layers it should not be required. Since this type of fixation is used for a long time there are plenty of well worked out protocols (including the best buffer for your system, the following dehydration steps in alcohol etc.) that may be worth to do a literature search for. :-)

Good luck.

I use 16% formaldehyde (methanol free), sold in glass ampules, 10 mL per ampule. It is available from several vendors. Unopened it can be stored at RT for long time. We dilute it with 1x PBS to make 4%. That diluted solution srored in 4oC for two weeks and then discarded if not used.

We usually fix cells at RT for 10-20 min but that would depend on your particular assay.

In the past, I have used 1-2% chilled paraformaldehyde. However, when I looked at my staining, the images weren't as nice. I have switched to using 100% MeOH fixation. I work with adherent cells that I place into PBS right before adding my MeOH, which I store at -20. As an example, if my coverslip with cells is covered in 1ml of PBS, then I add 1ml of 100% MeOH to each sample for a final concentration of 50% MeOH. Then I continue to add 1ml of MeOH to each sample, so that the final concentration ends up close to 100% MeOH. (Usually about 4 ml MeOH/per 1ml of PBS) I remove all the MeOH/PBS mixture and then place 100% ice cold MeOH on my cells for 10 mins. Then add 2ml of PBS to the MeOH and store the samples for further staining. I am attaching a PDF from Abcam about cell fixation and permeabilization for IHC/ICC samples. Hope this helps.

Hypotonic solution (tweak for your cell line), 4% paraformaldehyde chilled (stored in 4oC) for 10mins, and Triton 100-X for 5 mins, standard in our lab with good results. Best of luck

Hi, Florence, fixation at 4°C is not an optimal decision. I would suggest to use 2 fixation step: low temperature (4-10°C) for penetration and 37 °C for fixation. At 4°C formaldehyde have a tendency to polymerased and does not work so effective. There is a very nice recent publication about effect of temperature ion fixation http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0054138#pone-0054138-g015

Good luck!,

Hi Florence,

I agree with Taras, I uasually use 4% PFA in PBS and store it in -20 degrees for prolong usage. Since it is a highly cross-lonking agent and toxic, use it in a fume hood. Use low temperature for your fixation step, because room temperature will render harsh conditions.

Hi I am an undergrad working with chondrocytes. I noticed you mentioned coating slides with collagen. Could you let me know how the slide is prepared and whether this involves cells regards Gill