Fixing GFP strain for confocal microscopy

Hey :slight_smile:
I have a GFP strain, and I want to use the confocal to visualize the expression. I just wanted to know is there a protocol for fixing slides in such a way that it will not affect the GFP expression?
(The only reason I want to fix is to prevent my slides from dehydrating because the scope is another building on campus and I can’t prepare individual slides freshly before use).

Any suggestions? :smiley:

Thank you!

Can you mount the worms using levamisole or muscimol on pads in the other building? Alternatively, worms can keep on pads for a bit, say 30 minutes… how far away is the building?


We keep our slides in a ‘humidity’ chamber if we are going to image them in 30m-1h.

We have small plastic trays with lids (one similar to staining an agarose gel, for example. We use these from VWR.) We add a paper towel or filter paper to the bottom, saturate with water, then put parafilm on top. We also tape paper towel/filter paper to the lid and make that damp. The slide is kept safe from the water via the parafilm, but because it’s in a humid environment doesn’t easily dry out.

If you do this, definitely paralyze with levamisole vs azide, as azide will start to induce degeneration of tissues.

If you really want to stain, I’d recommend different protocols if you are imaging embryos or larvae. If embryos, freeze/crack with MeOH should preserve GFP (or you could stain with an anti-GFP antibody and use a secondary conjugated to something that fluoresces at 488, such as FITC or Cy2 or Alexa488). If larvae the Finney-Ruvkun formaldehyde/methanol fixation has preserved GFP in my hands.

Thank you very much. I will definitely be trying these suggestions this weekend. It was very helpful:)