Charles- I routinely use this method listed below. Which clathrin antibody are you using? If it is the BD clathrin heavy chain, I use it at 1:1000. Sometimes vesicles like to be fixed in warm PFA. Don't use glutaraldehyde- although it's great for some things, and is the preferred method for super res applications, it often masks epitope binding sites.

1) Warm reagents to room temperature:

- 4% PFA fresh from vial (EM grade), dilute16% PFA in BRB80 (see below for recipe, we routinely make a 5x stock and store it in the fridge).

-Make 0.25%TX-100 PBS (or TBS) for permeabilisation.

- AbDIL, for blocking make a bottle, filter sterilize it and add 0.02% NaAzide keeps in the fridge. Check routinely for “floaties” (see below for recipe)

4) PBS or TBS for washes

Remove cells from incubator, tilt dish and aspirate media from the side

Add 1ml-2ml PBS to the side of each well whilst the dish is tilted and allow to move over cells by placing dish on the bench flat

I’ve excluded this step many times and seen no difference, some people say ice cold PBS to stop trafficking etc, and again, I’ve seen no difference. I think they are more likely to retract upon the addition of cold buffers and prefer to fix immediately

Remove PBS and add 1ml of fixative for 10-20 minutes at room temp.

Wash 3-5 times with PBS, room temperature, 1-2 ml each to remove the fixative

Permeabilize cells by adding 1-2 ml 0.25% tx100/PBS for 5 minutes at room temp

Wash 3-5 times in 1-2 ml of PBS to remove traces of detergent (stop it putting too many holes in the membrane).

Add 1-2 ml AbDIL to each well, cover with foil and incubate overnight at 4C.

The next morning, arrange coverslips on a prepared humidity chamber (Nunc, Bioassay chambers make great IF humidity chambers). I usually place a piece of parafilm into a chamber and surround it with damp kim wipes. If you have a lot of cover slips, add a drop of Abdil to each as you place it in the chamber to prevent it drying out.

it is ideal to arrange samples correlating with antibody stains and cell type/condition

Dilute primary antibodies in abdil, add 25ul per coverslip (I’ve used 15 ul for precious antibodies before), incubate for an hour. Wash 3-5 times for 5 minutes each -can be done with PBS or Abdil (you can never over wash a sample, but you can under wash it) this step removes unbound ab

Dilute secondaries in AbDIL, (I commonly use 1:500 for alexa dyes, and they are fine). Add 25ul to each 15mm coverslip and incubate for 30-45 minutes. This is a good time to label your glass slides which you are going to mount your coverslips onto. I clean them with ethanol and a kim wipe. Sometimes glass shards are present from the factory.

Wash coverslips 3-5 times, 5 minutes each, in PBS.

Mount in mowiol 4-7ul/cs (this hardens overnight- if you are going to do super res or want to prevent volume artefacts, I recommend a glycerol based mounting media).

Place in a closed drawer to dry over night (it hardens like nail polish).Don’t freek out if you do it on a Friday and forget to put them away until Monday. Not ideal, but they survive.

Store at 4C in a box/slider holder

BRB80 (1X):

80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with

KOH tablets (we generally made as a 5X stock and stored at 4C)

AbDil

2% BSA

0.1%tx100

0.02%NaAzide (filter and keep at 4C)

make up in TBS

1.7%. Light fixation is the key to retaining antigenicity. Not all epitopes are as hardy as others, so to much fixative and spoil things for your antibody. Trade-off might be that you lose some integrity in your cell or tissue sample. Paraformaldehyde, I think, is the dry form of formaldehyde. Generally, you want the pre made stuff that comes sealed in a glass vial when the quality of the fixation counts.

FA is a very small molecule. When you add 2% you give the cells an osmotic shock of about 1Osm, when you double the concentration you give a shocj that is twice as high. For the fixation process it is probably not so relevant as 2% is allready an excess. But that depends a bit on the item that you want to fix.

Rate of diffusion will be a higher then. Again light fixation is the key. Time is an element in this. Of course the practice is an empirical one. Do what works best, like determining the right dilution of primary and secondary antibody for a western.

Don't know what you are working with, but this might prove helpful. see page 588. Guide to Yeast Genetics and Molecular and Cell Biology edited by Christine Guthrie, Gerald R. Immunofluorescence Methods of Yeast.

You haven't said what you're fixing. Cells in suspension fix much more rapidly and uniformly than organized tissues, and elevated FA concentrations can actually slow diffusion of FA into deeper areas of tissue by fixing a "shell" of material around the deeper cells, so those are considerations, too. With regard to loss of antigenicity, that almost always must be determined empirically. However, for fixed-cell IF we use as a standard procedure the method of (Tonkin, CJ et al. 2004. Molec. Biochem. Parasitol. 137: 13-27), which uses a fixative containing 4.0% FA + 0.0075% glutaraldehyde, and 0.1% TX-100 for permeabilization for antibody access. We've had to modify it slightly for use with Babesia, to include 0.012% glutaraldehyde but only 0.01% NP-40, as our target behaves slightly differently from Plasmodium. We have yet to run into any problems of lost antigenicity with any antibodies we've tried, even with the glutaraldehyde present, and it is usually worse for destroying antigenicity. So, a long answer to say it probably won't make much difference to antigenicity, and what may be more important is overall structural preservation.

Firstly I make a 'standard' fixative solution from 4% paraformaldehyde in phosphate buffered saline (PBS) by measuring out the paraformaldehyde into the appropriate amount of PBS and rolling at 37ºC O/N. I aliquot this out and freeze it. I then dilute this by half (again in PBS) if I am using it as a 2% solution. These concentrations are important.

As has been said, the level of fixation, while increasing the physical stability of the fixed tissue by maximising the amount of covalent cross-linked chemical bonds, reduces the level of epitopes available for your primary antibody to bind to; therefore (in my simplistic view) Ab binding (the eventual 'signal') to fixation levels are inversely proportional. Lighter fixations, while increasing the chance of Ab binding, means that the integrity of the tissue is compromised and if you are doing long term incubations and/or washes, or the tissues themselves are very fragile, then the tissues can/will degrade.

Again the level of fixation can be varied by the time spent incubating and the temperature. For me, these conditions range between 10 - 30 mins for time and between 4ºC (ie. on ice) to RT for the temperature (always with gentle rocking). Again the longer you incubate with fix and the higher the temperature the greater the level of fixation will occur (greater number of covalent cross-linked chemical bonds) and the less chance of the primary Ab binding.

However sometimes the lightest fixation is absolutely necessary. In my case I have had to (successfully, though it is a pain) fix Drosophila pupal CNSs in 2% fix for 10-20 mins on ice in order to achieve a return of signal with respect to immunofluorescence.

There are other weird and wonderful fixations that can be performed (using Gluteraldehydes and incorporating Picric Acid) etc. but these are specific to the samples and Abs employed. Also while heat and/or pressure treatments for antigenic retrieval (required when the tissue is so fixed that no signal is achieved due to the lack of binding of the Ab) usually is employed on far more robust tissues, I do know colleagues who have successfully employed (light) heat treatment to Drosophila tissues for antigenic retrieval .

Finally It should be noted that the Paraformaldehyde itself is not a fixative, rather it is the higher polymer (n up to 100), stable (relatively insoluble) white powder that I make the fixative from. It must be depolymerized so that the solution contains monomeric formaldehyde (aka methylene hydrate) as its major solute to be useful as a fixative, . Dilution with water breaks up the small polymers into formalin.

Hope this is of some help.

The paraformaldehyde (PFA) is polymerized form of formaldehyde (solid form). It is used for fixation after dissolving in water under heating. PFA is methanol free and is preferred to use for fixation if contaminant free solution is needed. The commercially available aqueous solution of formaldehyde often contain methanol which is used to prevent spontaneous polymerization of formaldehyde.

In general choosing fixation method depends on what type of structures you are looking at, as at the resolution level of classical wide field microscope for example, the damage on the sample (due to fixation) is not easily observed. However unsuitable fixation could alter the antibody-binding epitope on a protein and may affect the antibody’s binding affinity.

Glutaraldehy is highly fluorescent and absorbs. GA fixation might increase your autofluorescence or cause an inner filter effect. We use 0.025% GA and 2.7% FA mix (but this is for bacteria in suspension and as allready said by the various authors, fixation depends on the item you want to fix)

Thank you for your responses so far. I am trying to fix Hela Cells and stain for clathrin.

usually cells from culture are well fixed with 3.7% formaldehyde (also called formalin), it is easy to make because stock solution are ... 37% dont forget PBS. I never heard that this could be used to fix tissue or whole animals by perfusion

paraformaldehyde is generally used to fix tissue tissues 4% or 2% depending on a lot of parameters.

In some cases these fixations prevent antibody recognition and a different fixation has to be used, like methanol ole histology manuals are probably the best source of information about how to fix what kind of structures.

Paraformaldehyde is the powder/solid form. Fixation with formaldehyde made fresh by solubilizing paraformaldehyde is preferable. We typically will make 8% formaldehyde, titrate to neutrality with KOH and then mix with equal volume of 2X DMEM. The concentration of formaldehyde used should match your antibody profiles.. 2-4% formaldehyde is usually fine for antibody-based localization studies on cultured cells

Charles- I routinely use this method listed below. Which clathrin antibody are you using? If it is the BD clathrin heavy chain, I use it at 1:1000. Sometimes vesicles like to be fixed in warm PFA. Don't use glutaraldehyde- although it's great for some things, and is the preferred method for super res applications, it often masks epitope binding sites.

1) Warm reagents to room temperature:

- 4% PFA fresh from vial (EM grade), dilute16% PFA in BRB80 (see below for recipe, we routinely make a 5x stock and store it in the fridge).

-Make 0.25%TX-100 PBS (or TBS) for permeabilisation.

- AbDIL, for blocking make a bottle, filter sterilize it and add 0.02% NaAzide keeps in the fridge. Check routinely for “floaties” (see below for recipe)

4) PBS or TBS for washes

Remove cells from incubator, tilt dish and aspirate media from the side

Add 1ml-2ml PBS to the side of each well whilst the dish is tilted and allow to move over cells by placing dish on the bench flat

I’ve excluded this step many times and seen no difference, some people say ice cold PBS to stop trafficking etc, and again, I’ve seen no difference. I think they are more likely to retract upon the addition of cold buffers and prefer to fix immediately

Remove PBS and add 1ml of fixative for 10-20 minutes at room temp.

Wash 3-5 times with PBS, room temperature, 1-2 ml each to remove the fixative

Permeabilize cells by adding 1-2 ml 0.25% tx100/PBS for 5 minutes at room temp

Wash 3-5 times in 1-2 ml of PBS to remove traces of detergent (stop it putting too many holes in the membrane).

Add 1-2 ml AbDIL to each well, cover with foil and incubate overnight at 4C.

The next morning, arrange coverslips on a prepared humidity chamber (Nunc, Bioassay chambers make great IF humidity chambers). I usually place a piece of parafilm into a chamber and surround it with damp kim wipes. If you have a lot of cover slips, add a drop of Abdil to each as you place it in the chamber to prevent it drying out.

it is ideal to arrange samples correlating with antibody stains and cell type/condition

Dilute primary antibodies in abdil, add 25ul per coverslip (I’ve used 15 ul for precious antibodies before), incubate for an hour. Wash 3-5 times for 5 minutes each -can be done with PBS or Abdil (you can never over wash a sample, but you can under wash it) this step removes unbound ab

Dilute secondaries in AbDIL, (I commonly use 1:500 for alexa dyes, and they are fine). Add 25ul to each 15mm coverslip and incubate for 30-45 minutes. This is a good time to label your glass slides which you are going to mount your coverslips onto. I clean them with ethanol and a kim wipe. Sometimes glass shards are present from the factory.

Wash coverslips 3-5 times, 5 minutes each, in PBS.

Mount in mowiol 4-7ul/cs (this hardens overnight- if you are going to do super res or want to prevent volume artefacts, I recommend a glycerol based mounting media).

Place in a closed drawer to dry over night (it hardens like nail polish).Don’t freek out if you do it on a Friday and forget to put them away until Monday. Not ideal, but they survive.

Store at 4C in a box/slider holder

BRB80 (1X):

80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with

KOH tablets (we generally made as a 5X stock and stored at 4C)

AbDil

2% BSA

0.1%tx100

0.02%NaAzide (filter and keep at 4C)

make up in TBS

It depends on tissue/ cell type you use. 2% formaldehyde is probably enough for a cell culture.

Should not be a big difference up to 10% concentration but as higher the concentration the more expensive is your study and the more harmful it for you. Take care!!!

Dear Charles, undoubtedly, for my experience, 4% paraformaldehyde is a good fixative. To preserve very good morphology I suggests to you PLP fixative that generally I prepare mixing immediately before the use equal volume of 8% (w/v) paraformaldehyde in bidistilled water and 0.04 mol/L lysine in twice concentrated (i.e. 0.2mol/L) PBS and adding 0,55g/L NaIO4. Regards Stefano

Dear Charles, I have experienced working with 3% paraformaldehyde, and it is a good fixative for cells from culture for preserving immunofluorescence staining to flow cytometric analysis.

Hi Charles, i used 4%PFA by removed a half volume of media and added a half of 4% PFA as same as media volume. Then i incubated cell culture plates in 37c 20min. That was fine.

I washed coverglasses in pbs 3 times, after that, the formalin residues was removed by washing in 50mM NH4Cl.

HeLa cells can be fixed by treating with 2 or 2.5% PFA (diluted in warm PBS or PB) for 20 min. PFA cross links with cells proteins (means most of the cytoskeleton).

If one want to store cells for longer before imaging, can do so by keeping them in 1% PFA but remember to wash away PFA at least twice with PBS before imaging. That is what we do for confocal/photothermal imaging.

The difference between 2% and 3.7% is not very dramatic in my hands, assuming you are fixing for 15-20 minutes. However, formalin contains a small amount of methanol as a stabilizer, which will permeabilize some cells--not what you want if you intend to stain for cell-surface antigens. If you are staining for intracellular antigens, how you permeabilize AFTER fixation can also dramatically affect your results. Detergents tend to discriminate against cytoskeletal elements but allow better access of antibodies to the nucleus; post-fixation with -20 methanol (after PFA) allows better retention of cytoskeletal elements but removed lipid-anchored proteins. It's actually pretty complicated-- you can download a detailed protocol and notes from http://confocal.bwh.harvard.edu/guides.html under "How to fix, stain, permeabilize, and more for optimal images!"

Good luck!

Nancy is correct about the methanol used in commercial preparations. If this becomes an issue then always best to prepare fresh as described above from paraformaldehyde (powder) and dilute to around 2% w/v in phosphate buffered saline. The PLP fix is a classic approach to better preserve carbohydrate on cell surface. If the cytoskeleton is your target you can actually get some nice results permeabilising first (eg 0.1% Triton x100) in a low calcium buffer designed to preserve micro tubules. Rhodamine phalloidin can also be added at same time to stain and stabilise actin filaments (see Harkin DG, Hay ED. Cell Motil Cytoskeleton. 1996;35(4):345-57.

A comment I didn't think to add earlier is that, with regard to loss of antigenicity after fixation, monoclonal primary antibodies are more likely to be affected than are polyclonal antibodies because they recognize a single epitope. Polyclonal antibodies, with recognition of multiple epitopes, are more likely to still find unmodified epitopes (and the epitopes they recognize will have differing sensitivities to fixation). So, if antigenicity loss is a problem and you have more than one choice of antibody, you're likely to be better off with a good polyclonal preparation as primary antibody than with a monoclonal. Good luck.

Practically all antibodies to cytoskeletal proteins worked better after 1-2%PFA fixation (15 min) in warm culture medium without serum compared to 3-3,7%PFA, bur cold MeOH 5' after PFA. Just try: it is dependent on your antigen.

Charles,

There is a distinction to be made between PFA, formaldehyde and formalin. While you will see different people use the terms differently, I believe that the general use of the terms is as follows. As you may know, PFA is a pure form made from powder (or purchased in small vials kept cold and unstable) and many scientist swear that this is what you want for immunolabeling. Formaldehyde often refers to a diluted solution of 37% formaldehyde typically kept at room temperature. This is most often used for routine histology fixation and coloration. It is sometime frowned upon to use this for immunolabeling because it contains many impurities and degradation products, including formic acid. That being said, I have used it many times with good results; it depends on the antibody and antigen (and goal). Finally, formalin is an archaic term that comes from pathology labs. It also refers to a dilution of the concentrated 37% formaldehyde but considers the initial concentration to be 100%. For example, the popular 10% formalin is a 10X dilution of the 37% formaldehyde concentrate, i.e. 3.7% formaldehyde.

There are not a big differences between use mentioned above concentrations of PFA for a fixation, but you have to prolong time of fixation of tissue or cells with 2% of PFA

Haven't seen much difference between using 2% or 4% PFA, but for some stainings it is very important that it is freshly made if you make the solution yourself.

I am trying to see very specific co-localization on the plasma membrane. Has anyone ever seen co-localization of clathrin on the membrane using the X22 antibody with tight junctions or gap junctions?

How are you visualizing? If you're using confocal you may be ok. If you're doing standard epifluorescence you'll probably need to perform deconvolution of z-stacks of the images to have confidence in co-localization. If you need to do the latter, the "Parallel Iterative Deconvolution" algorithm available for ImageJ does a great job, and it's free. We do co-localization all the time of parasite proteins on the erythrocyte membrane surface under oil immersion, using a 100x NA 1.30 lens and deconvolution of the images. You will have the added difficulty of looking at junctions that are oriented perpendicular to your plane of view, but you may find that higher magnification, with its limited depth-of-field and deconvolution will provide just what you need. Good luck.

Why did we ever made handbooks for fixation and staining in histology and cell biology. Please look into e.g.M.A.Hyat' s handbooks (2002) or Humason's Animal tissue techniques (1972), old but very useful. There you will find that formaldehyde mainly contains 99% methylene glycol. It is the methylene glycol that penetrates fastly the tissue. Paraformaldehyde is of another dimension. There is enough in handbooks to answer your question thoroughly and fastly. There are also series of handbooks for immunostaining on light microscopic and electron microscopic sections. Sorry, I feel old!

M.A.Hayat: Microscopy, immunohistochemistry and antigen retrieval methods for light and electron microscopy. Kluwer Academic/Plenum Publishers, NY, Boston, Dordrecht, London, Moscow ISBN 0-306-46770-4 see chap 3 pp 53-60

Sorry for the late answer, was abroad.