Hi everyone, Part of my PhD study is to determine the presence of SR-B1 protein after treatment of selected compound in animal model (rat). Liver was cut into small pieces and soaked in 4% para-formaldehyde for 2 hours and 30% of sucrose for 24 hours. After the incubation, the liver was mounted on a stub and preserved with tissue freezing medium to make it easier to cut by using cryostat. The liver block then was cut into thin slice of 5 micron and put on a slide for fixing and staining with antibody. We viewed the slides under confocal microscope (40x magnification). After the staining, the results obtained seem to differ from what we expected earlier. The nucleus (blue, stained with dapi) showed irregular form while the fitc (green) stained the entire cell. The cells also showed "gaps" between cell (shrink?). I attached the image below to give a better view of the result.
We postulated that the incubation with 30% sucrose for 24 hours shrunk and altered the normal appearance of the cells. From an article that we referred, fitc will stain only a small part of the cell because SR-B1 only presence not at the entire cell surface and the result that we obtained shows contradiction with it. I hope there are comments that can help us to justify the results as well as to troubleshoot (if any) on the experimental procedure. Luke
Hi Luqman, did you use buffered 4% PFA? You could perform a simple hematoxylin-eosin staining to one of your slides in order to check tissue after fixation and cryopreservation. Did the same phenomena occur in your control livers?
Hi Luke,
How do you freeze your tissue: liquid nitrogen, -80°C, ...?
Did you wash well your liver pieces in PBS before fixation in order to remove the maximum of blood cells? (that can explain the strange nuclei you observed)
I'm not a specialist of liver, but 2h in 4% PFA is maybe not enough, you can try overnight incubation in PFA at 4°C then 8h in 15% sucrose and 8h in sucrose 30% (at 4°C too).
I agree with Flavie that 2h is quite a short fixation time for tissue pieces, unless they have been cut really small. Remember your fixative will penetrate by diffusion and its time will be limited by the radius of the piece as Fick's law states. Perhaps you could perfuse your mice with saline in advance in order to remove blood cells from capillaries. However, as the number of strange nuclear figures is big I insist in a simple histological staining to check your tissue.
Hi Flavie and Daniel,
Thanks for your suggestions!
Daniel:
yes i used buffered PFA.
Flavie:
I kept my sample in -80°C upon use and befor that the sample was washed with buffered formalin to eliminate excessive blood from the organ.
I'll come back with the results soon :)
Luke
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