Not all antibodies are created equal in terms of IF signal. Sigma's 8d4 is particularly good for talin. I've used 8d4 for detecting talin by IF in PFA-fixed samples in several publications. Make sure you also have a robust blocking buffer (such as 10% normal goat or normal donkey serum) to prevent non-specific adsorption of your primary and secondary antibodies.

In addition, staining for focal adhesion proteins works best if you remove the non-assembled cytosolic fraction of these proteins. The simplest way to achieve this is to perform simultaneous permeabilization/fixation using 4%PFA supplemented with 0.1% Triton X-100. Fix in this condition for 5' and follow with additional 15' of 4% PFA (no Triton). It is possible that the cytosolic fraction of talin is obscuring your focal adhesion signal.

Try freezing cold (keep methanol in -20 for an hour before fixing cells) methanol overnight at -20

Hi Paul

After washing media, try to fix cells with equal volume of methanol/PBS for 10 minutes and then fix cells in pure methanol for another 10 minutes.

Try using EM grade PFA (EMS Sells 16% solutions- throw them out after a week at -4c), diluted in BRB80 (not PBS).

Alternatively, try another Talin antibody.

BRB80 (1X):

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

KOH tablets (I generally make a 5X stock and store at 4C).

Hi Goodwin, I tried -20 methanol overnight with poor results unfortunately.

Gaber, what temperature should the two fixation steps be done at?

Thanks for that Samantha, would you dilute the PFA to 4% or 2% in BRB80 for this? I've seen people suggest varying concentrations for this type of fixation. And is this done at room temperature for 10 mins? Or 37C? I have tried two Talin antibodies

Thanks for your replies,

Paul

make my PFA 4%, and use at room temperature for 15-20 min.

wash 3-5 times in PBS (5-10 min)

Then permeabilise with 0.25% tx100, diluted in PBS for 5 minutes.

Followed by 3-5 x 5-10 min washes in PBS (room temp).

I block over night in ABDIL (2% BSA, 0.1%tx100, 0.02%NaAzide, filter and keep at 4C)

and primary for 1 hr (antibody diluted in ABDIL)

The Geiger lab should have some conditions where they fixed and stained for Talin- you could check out which antibodies they used?

I had similar issues trying to image microtubules with vinculin for a super-resolution project that required perfect fixation (ideally gluteraldehyde), which unfortunately destroyed the vinculin epitope, and the antibody no longer worked. I had to use several antibodies and fixation conditions to get one that worked.

Not all antibodies are created equal in terms of IF signal. Sigma's 8d4 is particularly good for talin. I've used 8d4 for detecting talin by IF in PFA-fixed samples in several publications. Make sure you also have a robust blocking buffer (such as 10% normal goat or normal donkey serum) to prevent non-specific adsorption of your primary and secondary antibodies.

In addition, staining for focal adhesion proteins works best if you remove the non-assembled cytosolic fraction of these proteins. The simplest way to achieve this is to perform simultaneous permeabilization/fixation using 4%PFA supplemented with 0.1% Triton X-100. Fix in this condition for 5' and follow with additional 15' of 4% PFA (no Triton). It is possible that the cytosolic fraction of talin is obscuring your focal adhesion signal.

I second Dan's suggestions about checking your antibody source to make sure it is appropriate for IF and having a good blocking protocol. Couple of additional thoughts - I noticed that you have a wash step with PBS prior to fixation. That could be avoided, especially if the PBS has no Ca/Mg in it, since it can weaken adhesions. Also, many of the reactions forming focal adhesions are labile - and the time out of the incubator and before fixation could be critical for the quality of the signal you see. I would add warmed paraformaldehyde (PFA) typically within a minute of taking cells out of the incubator.

I did not worked with talin in IF, but for vinculin, paxillin, FAK (F-actin as well) we have universal fixative: without prior PBS washing, 4% PFA+ 0.3% TritonX100 in PBS or any other suitable buffer for 10 min at RT (warm up your fixative though), wash 3x10 min PBS, block 1%BSA/PBS - 1 hr. The blocking solution may/may not have 5% normal serum of corresponding secondary antibody host. But if secondary were highly cross-absorbed against other spieces, it is not nessesary.

I know this is years later, but did you ever figure this out? I am also trying to stain for talin and vinculin and having problems. 

I use cytoskeleteal buffer instead of PBS for washing and preparing Blocking buffer.we do preferably fixative, permeabilizing, and blocking steps on ice not at room temperature.FIRST Aspirate media wash with PBS. prepare 4%PFA and 0.5% triton x and mix in 1:1 ratio (equal volume).

On ice add 1:1 solution and keep it for 1 min.

wash thrice with cold CSB.

add 4% PFA and keep it for 5 min on ice.

wash thrice with CSB.

add 1.5% blocking(prepared BSA in CSB) on ice for 30 min.

incubate anti vinculin in blocking solution overnight at 4C.

wash again with CSB and incubate with secondary in blocking for 4 hr at 4C.

at last wash thrice with CSB and mount on mounting solution, observe under microscope.