I am establishing a polarized gastric-epithelial monolayer culture on transwell system for bacterial infection studies. I use NCI-N87 cells and I culture them by replacing medium on every alternative day for 21 days. Later, I confirmed the expression of ZO-1 on 100% methanol (-20C) fixed cells. However, I face the following issues during this process.
1. How to avoid membrane curling while I cut off the membrane from the insert to mount on glass slide?
2. When I used 4% formaldehyde as a fixative to stain cell surface proteins, I found few cells or small cell clusters lying over the tight monolayer.
3. Is it necessary to use 21 days grown cells for bacterial infection studies? Because I see many highly cited papers also have used lesser days grown cells.
How to overcome these technicalities?
Any help is highly appreciated.
Thank you in advance.
Curling of the membrane happened to me too, on my PET and PC inserts. It cant be helped, as the fixatives are affecting the inserts. What I did after 1' and 2' antibody incubation was that, I cut the inserts into small square pieces (length abt 5mm), transfer to a glass slide, mounted and cover with a coverslip. For each insert, I probably used abt 6 coverslips on 3 slides.
For your second question, you'll need to wash the fixatives w PBS before antibody incubation.
And lastly, I used a min of 3 days old cells to a max of 1 week old. I was working on brain endothelial cells.
Hi Nurul Adhwa Rahman,
I tried to cut membrane into 4 pieces, still I observe curling. Next time I will try to cut into 6 as you mentioned and see.
Yes I do wash 3 times after fixation before blocking, so please help me understand your answer to my second question.
Will update if I could improve. Thanks a lot.
I use 0,4µm membrane inserts from Corning too, and even with cutting to 8 pieces I noticed curling to some degree. If your problem is to find a good focal plane, I can recommend using extended depth of focus function (AxioVision, requires additional license) on z-stacks, it solved the issue for me.
Hi Michal Mastarlerz, Thank you for the input. I will definitely enquire about this focus function in our microscope core.
I have more to add to the above question, what if the inserts have an area of 0.3 cm2 and I have to process the tissue grown on it for histology?
Hi Payal,
Thank you for adding more questions and perspectives to the topic.
When you grow tissue on inserts, if the tissue can be taken out of the insert without disrupting the architecture, that's best.
Or else, the tissue can be fixed in the plate itself by adding formaldehyde, excised along with the membrane post fixation and can be sectioned for histology.
I hope this helps.
Thank you for you answer Sasikala, I am growing the tissue without a scaffold just with FNC coating and the membrane as I mentioned has an area of 0.3 cm squared, will it be a problem during histology as the membrane tends to curl?
I would suggest to fix the tissue in the plate itself, remove-off the excess membrane if possible and then sectioning.
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