hi
i have been doing immunofluorescence on transwell membrane inserts and have been getting suboptimal images. i fix and stain the cells in transwell insert, and when i mount them i cut the membrane off, place it on glass slide with cells facing up. I then drop a few mounting media on membrane and then a coverslip over it. the pore size is 0.4um and does not permit migration.
1) i have tried fluorescence micoscopes and confocal microscopes but both are giving suboptimal imagings. What kind of microscope is best for this?
2) I notice that after placing the membrane on glass slide, and coverslip over it. there are still some slight creases (uneven regions of membrane that kinda forms bulging upwards). This could explain why when im focusing on one cell, the other regions get out of focused. I was thinking of putting pressure on the coverslip to straighten out the membrane. But will this affect morphology of the fixed cells?
any tips for people who had used this technique before?
What kind of stain are you using?
Instead of cutting the well we take a z-stack of the well after staining with DAPI starting from top (cells that have not migrated yet) to bottom (cells that have migrated), and it works fine with us. You can also try focusing on top and bottom and take separate images.
Post experiment we give it 1X PBS wash and fix it with 4% PFA within the well itself (20 min). After that wash with PBS again and stain it with DAPI and then place the trans-well insert directly on top of a cover-glass and take z-stack at a relatively low magnification (20X using an Zeiss LSM780 confocal microscope). Also put some PBS in the insert well to prevent it from drying and you also have to keep checking for PBS spillage through poor in the coverslip and microscope while imaging (once in a while wipe with a tissue paper if it spills a little). After staining you can even store the insert in PBS for a couple of days.
Dear Iwan
1) You should use confocal imaging, otherwise the membrane support will obscure your image. Suppose you have an upright microscope, right?
2) When using CLSM, the distance between the cells and the coverslip is of great importance, the closer the cells are to the coverslip, the better the image (since the light intensity is inversely proportional to the square of the distance from flourophore to objective, and since your objective has a certain working distance). We normally cut the insert in tiny bits (we typically divide an insert into 4-6 pieces), place a coverslip on the table, place a piece of the insert on the coverslip with the cells facing downwards (facing the coverslip), and then add a bit of mounting medium (or PBS as Amlan describes) on the "back" of the insert. Then we turn the coverslip around , place it on a glass slide, seal with polish and make CLSM imaging. The transwell inserts tend to bulge, but if you have small bits, there is a fair chance that a part of the insert will be in close contact with the cover slip. Search for these regions before yo do the actual imaging, the closer the cells are to the cover slip, the brighter a signal you will get. The procedure can be tricky though, it is important to keep track of whats "up" and "down" regarding the insert bits..
Best wishes
This is why I do not like the inserts and always have been avoiding them! Even if you could image them with a confocal and a long working distance objective, you simply can not get high resolution images. Its quite frustrating, and in my view, you will spend a lot of time fixing problems and then will abandon it due to poor quality of images.
If you can explain what you are doing may be alternative to inserts can be suggested. Regards
hi salam kabir,
i need to use the transwell insert for my compression experiments. I am using a pore size of 0.4um that allows movement of media and oxygen but not the transmigration of cells. So basically i grew cells on inside of transwell insert, then place a 5mm thick agarose-DMEM cushion, and then a compression cup (has a weight) over the agarose cushion to simulate compression. so as you can see, i cannot add that much of media inside transwell insert because the media would be ejected out upon placing compression cup. so i have to have media below the insert to allow cells have access to media and nutrients.
i tried compression by applying the agarose cushion and compression cup over a coverslip that has cells grown on it. this coverslip is placed on bottom of 6 well plate (no inserts). The cells died probably due to suffocation.
i would be very happy to get more suggestions.
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