In whole mount preparations of the retina around postnatal day 6 appeared a layer of hyaloids vessel/inner limiting membrane (I guess this layer is it?) preventing the access to retinal ganglion cells (RGCs) for patch clamp experiments, only some RGCs are patchable. In later developmental stages this problem rises. The same problem appeared in Ca2+-imaging experiments, when retinae/RGCs are loaded with Fura2, this hyaloid vessel layer/inner limiting membrane prevents the loading of RGCs with the dye. How is it possible to avoid this problem? How to remove this layer without destroying the retina to get an good access to RGCs in more mature retinal whole mount preparations?
Hi René, basically you ll have to use a first pipette with positive pressure to "clean", to scratch, to make an entrance in the membrane in order to get access to GCs. Different techniques are used, and I m pretty sure that you could find some tutorial videos on the jove.com website!
@Antoine: Thanks, to take a cleaning pipette I used recently for patch experiments, but for Ca-imaging I need a large clean area to measure calcium waves. JOVE sounds good.
Yes, the JoVE article from Kofuji's lab is a good help, with collagenase / hyaluronidase treatment. We've tested it (with just the collagenase, as we didn't have hyaluronidase in stock), and that helped significantly to go through the ILM.
@Michael: This JOVE video I have not been before, I will try the enzymatic treatment, thanks.
You also have this one: Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry, from Ivanova et al. They use local injections into the tissue to label vessels with isolectin - I guess a similar approach could be used for calcium dyes.
One way to solve this problem is to approach with 2 electrodes close to the cell that you want to patch, slightly touch electrodes tip to the inner limiting membrane and pull to the opposite direction. Cells will pup-up with clean surface, something that you need to get good gigaseal. Hope this help.
Thanks for all suggestions, the most successful way to remove the inner limiting membrane in my Ca2+-imaging experiments seems to be the short treatment with Collagenase/Hyaluronidase.
Dear Li,
this protocol I found:
Combine 10000 Units Collagenase (Worthington Chemicals) with 83 mg Hyaluronidase (Worthington Chemicals) into 4.150 mL of extracellular solution. Store in 50 μL aliquots at -20°C Aliquots can be used for several weeks. When ready to treat the retina, dilute aliquot into 500 μL of bubbled extracellular solution.
Treatment of retina with enzyme mixture and mounting in recording chamber:
Dilute an aliquot of collagenase/hyaluronidase in 500 μl extracellular solution, place solution in a 35 mm Petri dish and transfer the retina.
Keep the retina-containing Petri dish, covered and in darkness, in a 95% O2/5% CO2 environment for 15 min with gentle agitation. This step aids in digesting any remaining vitreous and allows for easier access to the ganglion cell layer. Tip: for retinas from younger animals with less vitreous or if adverse effects are observed, time can be shortened to ~5 minutes.
In the literature it was published, that the properties of RGCs are changed due to the treatment - zorry, I can not remember the paper.
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