Re: GFP tissue preparation for confocal microscopy

Posted by Sylvie Le Guyader-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-tissue-preparation-for-confocal-microscopy-tp2680696p2691238.html

I totally agree with Steve: only bad PFA kills GFP. I have done a fair bit of imaging GFP in filopodia which retracts very fast if not fixed properly.

 

My understanding is that paraformaldehyde is a polymer of formaldehyde and is solid. Formaldehyde is a gas that readily dissolves in water.

In a formaldehyde solution, two things happen:

- Formaldehyde equilibrates between the air above the solution and the solution itself so every time you open your bottle, more formaldehyde leaves the solution. Thus 15mL tubes are a good way to store working stocks because the volume/surface ratio limits this process.

- The second thing that happens is that paraformaldehyde forms as a precipitate which is definitely a sign that your solution needs to be trashed (not down the sink of course!). Thus I think it is not a good idea to buy ‘4%PFA’ in big bottles as it is usually sold except maybe if you use it very fast or if you have applications where the quality of fixation is not critical.

 

I prepare an 8% stock the following way:

Under a fume hood, warm up 150mL of PBS to about 60ºC

Add 16g of PFA powder

Stir and keep warm to dissolve

Adjust the pH to 7.4 with concentrate NaOH (you should then obtain a clear solution)

Adjust the volume to 200mL with PBS

Aliquote in 10 mL in 15mL falcon tubes

Store the aliquots for up to several years (currently using a 2 years old stock) at -20º

The working stock can be stored at 4ºC for a couple of months at the most

I normally pre warm the amount of solution I need to 37ºC and keep the rest in the fridge

I add the warm 2x solution directly onto my cells covered in the same volume of their own overnight medium (or changed at least 30 min before sot that the cells are very happyJ)

5 min at 37ºC for a single layer culture is plenty

Mount and image within 24h as the autofluorescence will quickly show up against your signal. You can image later but your signal to noise ratio will worsen with time.

I find this method excellent for preserving filopodia. It’s also trouble free since there is no rinsing the cells or making fresh solution every day.

 

Good luck!

 

Med vänlig hälsning / Best regards

 

Sylvie

 

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Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

[phone]

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Bunnell
Sent: 23 April 2009 17:20
To: [hidden email]
Subject: Re: GFP tissue preparation for confocal microscopy

 

PFA does not kill GFP fluorescence.

BAD PFA kills GFP fluorescence.

We routinely use 1% PFA with GFP, YFP, and CFP, and have never had a problem so long as the PFA is fresh and pH’ed to between 7.0 and 7.4.

    -Steve



On 4/22/09 10:34 PM, "Young Jik Kwon" <[hidden email]> wrote:

All,

We are trying to take confocal micrographs of at tumor tissues that express GFP. What would be the sample preparation procedures? We tried cryosectioned samples but the cell morphology seems weird. We already know paraformaldehyde fixation kills GFP fluorescence. Any expert's advice?

Best,

Young

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