I have mouse lungs fixed in 4% paraformaldehyde and am unsure how to prepare them for confocal microscopy. I'm looking to do a quick check if the Alexa Fluor 680-labeled nanoparticles within the lungs are still fluorescent (these samples are a couple years old though). Do the lungs have to be embedded in paraffin first, or could I just slice the raw tissue and somehow mount it to a slide?
Thanks!
I would embed the tissue in OTC (optimal tissue cutting compound) in a tissue tek molds. After the tissue has been embedded in the OTC, place the mold in -20 to freeze slowly. Once the OTC has frozen you can store it long term in -80. Once you are ready to proceed with staining, use a microtome set at -20 and cut 5-8 micron slices onto slides. I would not embed the tissue in parrafin if you are trying to perform fluorescent microscopy since it autofluoresces to a greater degree than OTC.
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I would embed the tissue in OTC (optimal tissue cutting compound) in a tissue tek molds. After the tissue has been embedded in the OTC, place the mold in -20 to freeze slowly. Once the OTC has frozen you can store it long term in -80. Once you are ready to proceed with staining, use a microtome set at -20 and cut 5-8 micron slices onto slides. I would not embed the tissue in parrafin if you are trying to perform fluorescent microscopy since it autofluoresces to a greater degree than OTC.
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You can place samples in 10% sucrose in PBS or directly in Tissue Tec for some days at 4 degree abd then freese its slowly in cryotome in embedding solution (Tissue Tec which is the same OCT )!!
Hello Ariel.
I agree with Brandon - paraffin shows autofluorescence. I would also prefer cryosections.
But before you freeze the tissue it is essential to cryoconserve it in 30% sucrose in order to avoid watercrystal formation which could destroy your tissue. The tissue is soaked in sucrose as soon as it sinks to the bottom of the tube. Depending on the size of it, this could take some time.
After that you should freeze the tissue quickly in isopentane and store it at -80°C until you cut it, to avoid crystal formation. For cutting, the frozen tissue can be placed in a tissue tek mold and embedded in OCT in the cryostat. Avoid thawing.
You can also cut up to 20um slices. In a CLSM you can make Z-stacks. If you want to subsequently stain the slices I would recommend the use of coated glass slides for mounting (like superfrost or histobond glass slides) to avoid detachment of the slices.
Hope these suggestions are helpful.
Greetings
Every type of tiisue has its own unique problems to deal with that the investigator needs to overcome. So it would be best to subdivide the tissue (assuming there is enough) and do frozen sections as well as embedding in paraffin to see which, if either, gives minimal autofluorescence. Fixation in an aldehyde, while providing better detail adds an additional amount of fluorescence due to the aldehyde group. After making an assesment of both approaches you can then go to the literature which describes several approaches to reducing the background fluoescence.
how thick a section can be for confocal? I´m starting with technique
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